User:Cushman.s/sandbox

Key parameters of cell microencapsulation technology[edit | edit source]

The potential of using cell microencapsulation in successful clinical applications can be realized only if several requirements encountered during the development process are optimized such as the use of an appropriate biocompatible polymer to form the mechanically and chemically stable semi-permeable matrix, production of uniformly sized microcapsules, use of an appropriate immune-compatible polycations cross-linked to the encapsulation polymer to stabilized the capsules, selection of a suitable cell type depending on the situation.

• Provides 3D micro environment simulating the extracellular matrix

Biomaterials[edit | edit source]

The use of the best biomaterial depending on the application is crucial in the development of drug delivery systems and tissue engineering. The polymer alginate is very commonly used due to its early discovery, easy availability and low cost but other materials such as cellulose sulphate, collagen, chitosan, gelatin and agarose have also been employed.

Alginate[edit | edit source]

Several groups have extensively studied several natural and synthetic polymers with the goal of developing the most suitable biomaterial for cell microencapsulation.Extensive work has been done using alginates which are regarded as the most suitable biomaterials for cell microencapsulation due to their abundance, excellent biocompatibility and biodegradability properties. Alginate is a natural polymer which can be extracted from seaweed and bacteria with numerous compositions based on the isolation source.

Alginate is not free from all criticism. Some researchers believe that alginates with high-M content could produce an inflammatory response and an abnormal cell growthwhile some have demonstrated that alginate with high-G content lead to an even higher cell overgrowth and inflammatory reaction in vivo as compared to intermediate-G alginates. Even ultrapure alginates may contain endotoxins, and polyphenols which could compromise the biocompatibility of the resultant cell microcapsules. It has been shown that even though purification processes successfully lower endotoxin and polyphenol content in the processed alginate, it is difficult to lower the protein contentand the purification processes could in turn modify the properties of the biomaterial. Thus it is essential that an effective purification process is designed so as to remove all the contaminants from alginate before it can be successfully used in clinical applications.

Modification and functionalization of alginate[edit | edit source]

Researchers have also been able to develop alginate microcapsules with an altered form of alginate with enhanced biocompatibility and higher resistance to osmotic swelling. Another approach to increasing the biocompatibility of the membrane biomaterial is through surface modification of the capsules using peptide and protein molecules which in turn controls the proliferation and rate of differentiation of the encapsulated cells. One group that has been working extensively on coupling the amino acid sequence Arg-Gly-Asp (RGD) to alginate hydrogels demonstrated that the cell behavior can be controlled by the RGD density coupled on the alginate gels. Alginate microparticles loaded with myoblast cells and functionalized with RGD allowed control over the growth and differentiation of the loaded cells. Another vital factor that controls the use of cell microcapsules in clinical applications is the development of a suitable immune-compatible polycation to coat the otherwise highly porous alginate beads and thus impart stability and immune protection to the system. Poly-L-lysine is the most commonly used polycation but its low biocompatibility restricts the successful clinical use of these PLL formulated microcapsules which attract inflammatory cells thus inducing necrosis of the loaded cells. Studies have also shown that alginate-PLL-alginate (APA) microcapsules demonstrate low mechanical stability and short term durability. Thus several research groups have been looking for alternatives to PLL and have demonstrated promising results with poly-L-ornithine and poly(methylene-co-guanidine) hydrochloride by fabricating durable microcapsules with high and controlled mechanical strength for cell encapsulation.

Several groups have also investigated the use of chitosan which is a naturally derived polycation as a potential replacement for PLL to fabricate alginate-chitosan (AC) microcapsules for cell delivery applications. However, studies have also shown that the stability of this AC membrane is again limited and one group demonstrated that modification of this alginate-chitosan microcapsules with genipin, a naturally occurring iridoid glucosid from gardenia fruits, to form genipin cross-linked alginate-chitosan (GCAC) microcapsules could augment stability of the cell loaded microcapsules. Microphotographs of the alginate-chitosan (AC) microcapsules.

Collagen[edit | edit source]

Collagen, a major protein component of the ECM, provides support to tissues like skin, cartilage, bones, blood vessels and ligaments and is thus considered a model scaffold or matrix for tissue engineering due to its properties of biocompatibility, biodegradability and ability to promote cell binding. This ability allows chitosan to control distribution of cells inside the polymeric system. Thus, Type-I collagen obtained from animal tissues is now successfully being used commercially as tissue engineered biomaterial for multiple applications.Collagen has also been used in nerve repair and bladder engineering. Immunogenicity has limited the applications of collagen. Gelatin has been considered as an alternative for that reason.

Gelatin[edit | edit source]

Gelatin is prepared from the denaturation of collagen and many desirable properties such as biodegradability, biocompatibility, non-immunogenity in physiological environments, and easy processability make this polymer a good choice for tissue engineering applications. It is used in engineering tissues for the skin, bone and cartilage and is used commercially for skin replacements.

Chitosan[edit | edit source]

Chitosan is a polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It is derived from the N-deacetylation of chitin and has been used for several applications such as drug delivery, space-filling implants and in wound dressings. However, one drawback of this polymer is its weak mechanical properties and is thus often combined with other polymers such collagen to form a polymer with stronger mechanical properties for cell encapsulation applications.

Agarose[edit | edit source]

Agarose is a polysaccharide derived from seaweed used for nanoencapsulation of cells and the cell/agarose suspension can be modified to form microbeads by reducing the temperature during preparation. However, one drawback with the microbeads so obtained is the possibility of cellular protrusion through the polymeric matrix wall after formation of the capsules.

Cellulose Sulphate[edit | edit source]

Cellulose sulphate is derived from cotton and, once processed appropriately, can be used as a biocompatible base in which to suspend cells. When the poly-anionic cellulose sulphate solution is immersed in a second, poly-cationic solution (e.g. pDADMAC), a semi-permeable membrane is formed around the suspended cells as a result of gelation between the two poly-ions. Both mammalian cell lines and bacterial cells remain viable and continue to replicate within the capsule membrane in order to fill-out the capsule. As such, in contrast to some other encapsulation materials, the capsules can be used to grow cells and act as such like a mini-bioreactor. The biocompatible nature of the material has been demonstrated by observation during studies using the cell-filled capsules themselves for implantation as well as isolated capsule material. Capsules formed from cellulose sulphate have been successfully used, showing safety and efficacy, in clinical and pre-clinical trials in both humans and animals, primarily as anti-cancer treatments, but also exploring possible uses for gene therapy or antibody therapies. Using cellulose sulphate it has been possible to manufacture encapsulated cells as a pharmaceutical product at large scale and fulfilling Good Manufacturing Process (cGMP) standards. This was achieved by the company Austrianova in 2007.

Biocompatibility[edit | edit source]

The use of an ideal high quality biomaterial with the inherent properties of biocompatibility is the most crucial factor that governs the long term efficiency of this technology. An ideal biomaterial for cell encapsulation should be one that is totally biocompatible, does not trigger an immune response in the host and does not interfere with cell homeostasis so as to ensure high cell viability. However, one major limitation has been the inability to reproduce the different biomaterials and the requirements to obtain a better understanding of the chemistry and biofunctionality of the biomaterials and the microencapsulation system. Several studies demonstrate that surface modification of these cell containing microparticles allows control over the growth and cellular differentiation. of the encapsulated cells.

One study proposed the use of zeta potential which measures the electric charge of the microcapsule as a means to predict the interfacial reaction between microcapsule and the surrounding tissue and in turn the biocompatibility of the delivery system.

Microcapsule permeability[edit | edit source]

A fundamental criterion that must be established while developing any device with a semi-permeable membrane is to adjust the permeability of the device in terms of entry and exit of molecules. It is essential that the cell microcapsule is designed with uniform thickness and should have a control over both the rate of molecules entering the capsule necessary for cell viability and the rate of therapeutic products and waste material exiting the capsule membrane. Immunoprotection of the loaded cell is the key issue that must be kept in mind while working on the permeability of the encapsulation membrane as not only immune cells but also antibodies and cytokines should be prevented entry into the microcapsule which in fact depends on the pore size of the biomembrane.

It has been shown that since different cell types have different metabolic requirements, thus depending on the cell type encapsulated in the membrane the permeability of the membrane has to be optimized. Several groups have been dedicated towards the study of membrane permeability of cell microcapsules and although the role of permeability of certain essential elements like oxygen has been demonstrated, the permeability requirements of each cell type are yet to be determined.

• sodium citrate degradation

Mechanical strength and durability[edit | edit source]

It is essential that the microcapsules have adequate membrane strength (mechanical stability) to endure physical and osmotic stress such as during the exchange of nutrients and waste products. The microcapsules should be strong enough and should not rupture on implantation as this could lead to an immune rejection of the encapsulated cells.For instance, in the case of xenotransplantation, a tighter more stable membrane would be required in comparison to allotransplantation. Also, while investigating the potential of using APA microcapsules loaded with bile salt hydrolase (BSH) overproducing active Lactobacillus plantarum 80 cells, in a simulated gastro intestinal tract model for oral delivery applications, the mechanical integrity and shape of the microcapsules was evaluated. It was shown that APA microcapsules could potentially be used in the oral delivery of live bacterial cells. However, further research proved that the GCAC microcapsules possess a higher mechanical stability as compared to APA microcapsules for oral delivery applications. Martoni et al. were experimenting with bacteria-filled capsules that would be taken by mouth to reduce serum cholesterol. The capsules were pumped through a series of vessels simulating the human GI tract to determine how well the capsules would survive in the body. Extensive research into the mechanical properties of the biomaterial to be used for cell microencapsulation is necessary to determine the durability of the microcapsules during production and especially for in vivo applications where a sustained release of the therapeutic product over long durations is required.

Illustration of the APA microcapsule integrity and morphological changes during simulated GI transit. (a) Pre-stomach transit. (b) Post-stomach transit (60 minutes). (c) Post-stomach (60 minutes) and intestinal (10-hour) transit. Microcapsule size: (a) 608 ± 36 μm (b) 544 ± 40 μm (c) 725 ± 55 μm. From Martoni et al. (2007

Cryopreservation

• how durable under intensive freezing techniques

Methods for testing mechanical properties of microcapsules

• Rheometer
  • shear rate
  • shear strength
  • consistency coefficient
  • flow behavior index

• Viscometer - shear strength testing

Microcapsule Generation

Microfluidics

• manipulation of alginate solution to allow microcapsules to be created

Electrospraying Techniques

• Feeding alginate through a nozzle which contains a high voltage to usually a ground source or lower voltage
• Forms beads due to charge and surface tension
• Calcium Chloride used as cross linking solution (other solutions used for gelatin cross-linking)
• Size dependency
  • height alterations
  • voltage alterations
  • alginate concentration alterations

Microcapsule size[edit | edit source]

The diameter of the microcapsules is an important factor that influences both the immune response towards the cell microcapsules as well as the mass transport across the capsule membrane. Studies show that the cellular response to smaller capsules is much lesser as compared to larger capsules and in general the diameter of the cell loaded microcapsules should be between 350-450 µm so as to enable effective diffusion across the semi-permeable membrane.

Cell choice[edit | edit source]

The cell type chosen for this technique depends on the desired application of the cell microcapsules. The cells put into the capsules can be from the patient (autologous cells), from another donor (allogeneic cells) or from other species (xenogeneic cells). The use of autologous cells in microencapsulation therapy is limited by the availability of these cells and even though xenogeneic cells are easily accessible, danger of possible transmission of viruses, especially porcine endogenous retrovirus to the patient restricts their clinical application, and after much debate several groups have concluded that studies should involve the use of allogeneic instead of xenogeneic cells. Depending on the application, the cells can be genetically altered to express any required protein. However, enough research has to be carried out to validate the safety and stability of the expressed gene before these types of cells can be used.

This technology has not received approval for clinical trial because of the high immunogenicity of cells loaded in the capsules. They secrete cytokines and produce a severe inflammatory reaction at the implantation site around the capsules, in turn leading to a decrease in viability of the encapsulated cells. One promising approach being studied is the administration of anti-inflammatory drugs to reduce the immune response produced due to administration of the cell loaded microcapsules. Another approach which is now the focus of extensive research is the use of stem cells such as mesenchymal stem cells for long term cell microencapsulation and cell therapy applications in hopes of reducing the immune response in the patient after implantation. Another issue which compromises long term viability of the microencapsulated cells is the use of fast proliferating cell lines which eventually fill up the entire system and lead to decrease in the diffusion efficiency across the semi-permeable membrane of the capsule. A solution to this could be in the use of cell types such as myoblasts which do not proliferate after the microencapsulation procedure.

Volvox Spheres

• what volvox is
• inner beads within outer bead
  • size difference and alginate concentration differences
• basic protocol using electrospraying techniques and how to coat the inner bead before outer bead can be generated
• importance for micro environments
• for multiple cell types
  • growth/development in terms of resources
  • growth factors
• can be used for improving cell viability without having one cell type out grow and monopolize all the available resources
  • separation within inner beads for limited media/food sources
  • contained within an outer bead for a intimate microenvironment that still allows cell signaling contact and gas and nutrient exchange due to membrane permeability