Cell encapsulation

The first successful experiments in the direction of cell encapsulation in polymer membranes were published in 1934 by Vincenzo Biscelier ^[1] . He demonstrated that tumor cells in the polymer structure transplanted into the abdominal cavity of the pig remain viable for a long period without rejection by their immune system.

Thirty years later, in 1964, proposed the idea of ​​encapsulating cells in ultrathin membranes, and he coined the term to define the concept of bioencapsulation. He suggested that these capsules, obtained by the drip method, not only protect hidden cells from immune rejection, but also provide a high surface to volume ratio, which will increase the delivery of oxygen and nutrients. Twenty years later, this approach was successfully put into practice in small model animals when microcapsules from alginate-polylysine-alginate (APA) were developed for transplanted islet cells into diabetic rats. The study showed that the cells remained viable and controlled glucose levels for several weeks. In 1998, human trials began: encapsulated cells producing cytochrome P450 were successfully used in inoperable pancreatic cancer. The life extension of patients was approximately two times greater than previously known similar cases.

Encapsulated cells provide researchers and doctors with a number of additional features. First, such cells can release drugs for a long time at the site of their implantation. Such drug delivery methods are more accurate and economical than traditional methods. Secondly, it becomes possible to use animals and genetically modified cells in the event of a shortage of donor cells. Thirdly, artificial cells can be introduced to different patients, regardless of their leukocyte antigen, which reduces the cost of treatment.

The potential for using microencapsulated cells in successful clinical trials can be realized if requirements arising in the development process are met, such as using an appropriate biocompatible polymer that forms a mechanically and chemically stable semipermeable membrane; production of microcapsules of the same size; the use of appropriate immunocompatible polycations; selecting the appropriate cell type.

Biomaterials

The use of the best biomaterial depending on the application is crucial in the development of drug delivery systems and tissue engineering. Alginate is very widely used due to its availability and low cost, but other materials have also been used, such as cellulose, collagen sulfate, chitosan, gelatin and agarose.

Alginate

Several groups have studied in detail several natural and synthetic polymers in order to develop the most suitable biomaterial for microencapsulation cells. Natural alginate polymers are considered the most suitable materials for microencapsulation because of their accessibility, excellent biocompatibility and their ability to be easily biodegradable.

Alginate is not without flaws. Some researchers believe that high mannuronic acid alginate can cause an inflammatory reaction and abnormal cell growth. Others have shown that alginate with a high content of glucuronic acid leads to even more active cell growth and an inflammatory response in vivo. Even ultrapure alginates can contain endotoxins and polyphenols , which can compromise the biocompatibility of the resulting encapsulated cells. Purification of alginates reduces the content of endotoxins and polyphenols, but changes the properties of the biomaterial.

Alginate Modification and Functionalization

Researchers were also able to develop microcapsules with a modified form of alginate with increased biocompatibility and high resistance to osmotic swelling. Another approach to increasing the biocompatibility of the biomaterial membrane is to modify the surface of the capsule using peptide and protein molecules, which, in turn, control the proliferation and differentiation rate of encapsulated cells. One group that is actively working on linking the amino acid sequence of Arg-Gly-Asp (RGD) to alginate hydrogels has shown that cell behavior can be controlled using RGD density in combination with alginate gel. Alginate microparticles loaded with myoblasts cells and functionalized RGD allowed to control the growth and differentiation of loaded cells. Another important factor that controls the use of cell microcapsules in clinical practice is the development of a suitable immunocompatible polycation to cover, otherwise, the highly porous alginate granules and therefore impart stability and immune defense to the system. is the most widely used polycation, but its low biocompatibility limits the successful clinical use of these poly-L-lysine formulated microcapsules that attract inflammatory cells, thereby causing necrosis of the loaded cells. Studies have also shown that microcapsules alginate-P-L-L-alginate (APA) showed low mechanical stability and short life. Thus, several research groups have looked for alternatives to P-L-L and have shown promising results with poly-L-ornithine and poly (methylene hydrochloride-co-guanidine) for the manufacture of robust microcapsules with high and controlled mechanical strength for cell encapsulation. Several groups have also investigated the use of chitosan, which is the natural origin of the polycation, as a potential substitute for P-L-L in the manufacture of alginate-chitosans (A X) microcapsules for cell delivery programs. Nevertheless, studies also showed that the stability of the alginate-chitosan membrane is again limited, and one group showed that the modification of alginate-chitosan microcapsules with genipin (in nature it is iridoid glycoside from fruit gardenia), forming genipine cross-linking the alginate microcapsule - chitosan (HA ), improves the stability of the cell loaded microcapsules.

Gelatin

Gelatin is obtained by collagen denaturation. With many of the required properties, such as biodegradability, biocompatibility, non-immunogenicity under physiological conditions and easy processability, this polymer is a good choice for tissue engineering. Used in tissue engineering of the skin, bones and cartilage.

Chitosan

Chitosan is a polysaccharide consisting of randomly distributed monomer units of D-glucosamine and N-acetyl-D-glucosamine, connected by β- (1-4) bonds. It is obtained from N-deacetylation and partial hydrolysis of chitin, is actively being studied for the tasks of drug delivery systems (including targeted therapy), filling the space of implants, integumentary and dressings. The disadvantage of this polymer is its weak mechanical properties, however, it is successfully used to encapsulate cells in combination with other polymers, in particular, collagen.

Agarose

Agarose is a polysaccharide derived from seaweed used to nano-encapsulate cells and cells of an agarose suspension, which can be modified to form microgranules by lowering temperature during cooking. However, one of the disadvantages of the microgranules thus obtained is the possibility of cell access through the polymer matrix wall after capsule formation.

Cellulose Sulphate

Cellulose sulfate is obtained from cotton and, after proper treatment, can be used as a biocompatible base in which cell immobilization occurs. When a cell suspension in a polyanionic cellulose sulfate solution is added to a solution of another polycationic polymer (e.g. pDADMAC), a semipermeable membrane is formed around the suspended cells as a result of gelation between the two poly-ions. Like mammalian cells, the act and bacteria under such conditions remain viable and continue to replicate inside the membrane capsule. Thus, unlike some other encapsulation materials, this approach can be used to grow their cells as a mini-bioreactor. The biocompatible nature of the material was demonstrated in studies using cell-filled capsules for implantation, as well as capsules of isolated material ^{[ what? ]} . Cellulose sulfate capsules have successfully passed preclinical and clinical trials, both in animals and in humans, primarily for the treatment of tumors, but they are still being studied for possible other uses.

Biocompatibility

The use of ideal high-quality biomaterial with inherent biocompatibility properties is the most important factor that determines the long-term effectiveness of this technology. An ideal biomaterial for cell encapsulation should be one that is fully biocompatible and does not cause an immune response in the host, and does not interfere with cell homeostasis , such as to ensure high cell viability. However, one of the main limitations was the inability to reproduce various biomaterials and requirements in order to gain a better understanding of the chemistry and biofunctionality of biomaterials and the microcapsule system. Several studies have shown that surface modification of these cells containing microparticles allows control of the growth and cell differentiation of encapsulated cells. One study suggested using the zeta potential, which measures the electrical charge of a microcapsule as a means to predict interfacial interaction between the microcapsule and surrounding tissue, and in turn the biocompatibility of the delivery system.

• ChangTM Semipermeable microcapsules (Eng.) // Science. - October 1964. - Vol. 146, no. 3643 . - P. 524-525. - DOI : 10.1126 / science.146.3643.524 .
• Lim F., Sun AM Microencapsulated islets as bioartificial endocrine pancreas (Eng.) // Science. - November 1980. - Vol. 210, no. 4472 . - P. 908–910. - DOI : 10.1126 / science.6776628 .
• Löhr M., Bago ZT, Bergmeister H., Ceijna M., Freund M., Gelbmann W., Günzburg WH, Jesnowski R., Hain J., Hauenstein K., Henninger W., Hoffmeyer A., ​​Karle P., Kröger JC, Kundt G., Liebe S., Losert U., Müller P., Probst A., Püschel K., Renner M., Renz R., Saller R., Salmons B., Walter I. Cell therapy using microencapsulated 293 cells transfected with a gene construct expressing CYP2B1, an ifosfamide converting enzyme, instilled intra-arterially in patients with advanced-stage pancreatic carcinoma: a phase I / II study // Journal of molecular medicine (Berlin, Germany). - April 1999. - Vol. 77, no. 4 . - P. 393–398. - DOI : 10.1007 / s001090050366 .
• Löhr, M; Hoffmeyer, A; Kröger, J; Freund, M; Hain, J; Holle, A; Karle, P; Knöfel, WT; Liebe, S; Müller, P; Nizze, H; Renner, M; Saller, RM; Wagner, T; Hauenstein, K; Günzburg, WH; Salmons, B (May 19, 2001). "Microencapsulated cell-mediated treatment of inoperable pancreatic carcinoma.". Lancet 357 (9268): 1591–2. doi: 10.1016 / S0140-6736 (00) 04749-8 . PMID 11377651
• Lohr, M; Kroger, JC .; Hoffmeyer, A .; Freund, M .; Hain, J .; Holle, A .; Knofel, WT; Liebe, S .; Nizze, H .; Renner, M .; Saller, R .; Karle, P .; Muller, P .; Wagner, T .; Hauenstein, K .; Salmons, B .; Gunzberg, WH (2003). "Safety, feasibility and clinical benefit of localized chemotherapy using microencapsulated cells for inoperable pancreatic carcinoma in a phase I / II trial." Cancer Therapy 1 : 121–31.
• Murua A, Portero A, Orive G, Hernández RM, de Castro M, Pedraz JL (December 2008). "Cell microencapsulation technology: towards clinical application." J Control Release 132 (2): 76–83. doi: 10.1016 / j.jconrel.2008.08.010 . PMID 18789985 .
• Sakai S, Kawabata K, Ono T, Ijima H, Kawakami K (August 2005). "Development of mammalian cell-enclosing subsieve-size agarose capsules (<100 microm) for cell therapy." Biomaterials 26 (23): 4786–92. doi: 10.1016 / j.biomaterials.2004.11.043 .PMID 1576325
• Cellesi F, Weber W, Fussenegger M, Hubbell JA, Tirelli N (December 2004). "Towards a fully synthetic substitute of alginate: optimization of a thermal gelation / chemical cross-linking scheme (" tandem "gelation) for the production of beads and liquid-core capsules." Biotechnol. Bioeng. 88 (6): 740 doi: 10.1002 / bit.20264 .PMID 15532084 .
• Otterlei M, Ostgaard K, Skjåk-Braek G, Smidsrød O, Soon-Shiong P, Espevik T (August 1991). "Induction of cytokine production from human monocytes stimulated with alginate." J. Immunother. 10 (4): 286–91. doi: 10.1097 / 00002371-199108000-00007 .PMID 1931864 .