Encapsulation of Cells in Alginate Gels

The Revised Authoritative Guide To Vaccine Legal Exemptions

Vaccines Have Serious Side Effects

Get Instant Access

Gorka Orive, Rosa María Hernández, Alicia Rodríguez Gascón, and José Luis Pedraz

Summary

Cell encapsulation represents one of the current leading methodologies aimed at the delivery of biological products to patients for the treatment of multiple diseases. Alginate is the most frequently employed material for the elaboration of the polymer matrix and outer biocompatible membrane because of its mild gelling and biocompatibility and biode-gradability properties. However, its successful exploitation requires knowledge of microencapsulation technology and of the main properties of alginates, including their composition, purity, and viscosity. This chapter will focus on the immobilization of cells in alginate gels to create alginate-PLL-alginate microcapsules using an electrostatic droplet generator.

Key Words: Alginate; biocompatibility; biomaterial; cell encapsulation; microcapsules. 1. Introduction

Biomaterials are already having an enormous impact on medicine. Fabrication of pure and highly biocompatible polymers have allowed scientists to apply them in multiple scientific areas, including tissue engineering (1), dental implants, wound dressings (2), and drug delivery (3,4). In order to provide different alternatives for each specific approach, a large variety of natural and synthetic polymers are currently available in medicine. In particular, natural polymers are attractive biomaterials because of their abundance and apparent biocompatibility. One of the most frequently employed natural polymers is alginate. Because of its mild gelling and biocompatibility and biodegradability properties, alginate has long been used in the food and pharmaceutical industries (5). Interestingly, there has been an increasing interest in using alginate in a wide range of scientific fields such as oral vaccination (6), development of controlled-release systems for conventional drugs and peptides (7,8), and cell microencapsulation for the controlled and continuous delivery of therapeutic products (9).

Guluronic Acid

Fig. 1. Chemical structure of the alginates. G corresponds to guluronic acids, whereas M belongs to mannuronic blocks.

Guluronic acid

Fig. 1. Chemical structure of the alginates. G corresponds to guluronic acids, whereas M belongs to mannuronic blocks.

1.1. Alginates

Alginates are naturally occurring polysaccharides extracted from the brown seaweeds (Phaeophycea) found in the shallow waters of temperate zones. Although alginate was first prepared by Stanford and a pure isolation was afterwards achieved by Krefting (10), both in the 1880s, it was not until the 1930s that alginate was first exploited commercially. Different species of brown seaweeds, such as Laminaria hyperborea and Macrocystis pyrifera, are responsible for producing alginates with different chemical composition. In fact, as shown in Fig. 1, alginates are a family of unbranched binary copolymers of 164 linked P-D-mannuronic acid (M) and a-L-guluronic acid (G). The alginate monomers occur as regions made up exclusively of one unit of M blocks or G blocks or as alternating structures (MG blocks) (11).

Because alginates do not have any regular repeating unit, the polymer chain cannot be described by Bernoullian statistics. Therefore, apart from the monomer composition, the diad and triad sequences must be measured by nuclear magnetic resonance (NMR) techniques if a complete characterization of the polymer is intended (12).

Numerous studies have shown that the monomeric composition, the molecular sequential structure, and gel-forming kinetics of an alginate have significant impact on some of its properties, including stability, mechanical resistance, permeability, biodegradability, and swelling behavior. Interestingly, there is a disagreement in the literature about the possible effects of all these properties on the biocompatibility and immunological characteristics of alginates and alginate microcapsules (13-15). Overall, it seems reasonable that purification of the polymers is a fundamental prerequisite for obtaining materials with improved biocompatibility properties. This can be observed clearly if nonpurified and purified alginates are tested using a tumor necrosis factor (TNF)-a production assay. As shown in Fig. 2A, the production of TNF-a (a proinflammatory cytokine involved in multiple inflammatory signals) (16) was approximately 100 times higher in a nonpurified alginate comparing with purified alginates of different composition and viscosity. Assuming this, the implantation of capsules elaborated with alginates of different degrees of purity gives as a result a completely different in vivo inflammatory response as shown in Fig. 2B. The presence of a fibroblast

Fibrotic Encapsulation
g Non-pur Purified
Biodegradable Alginate Capsule

Fig. 2. (A) TNF-a secretion induced by different concentrations of a nonpurified and a purified low viscosity M-rich alginate (unpublished data). (B) Sections of alginate capsules recovered 1 mo after implantation. Left: nonfibrotic response; right: visible fibrotic response. Note the fibroblast overgrowth over the capsules (unpublished data).

Fig. 2. (A) TNF-a secretion induced by different concentrations of a nonpurified and a purified low viscosity M-rich alginate (unpublished data). (B) Sections of alginate capsules recovered 1 mo after implantation. Left: nonfibrotic response; right: visible fibrotic response. Note the fibroblast overgrowth over the capsules (unpublished data).

growth layer over the capsules could impair effective secretion of the therapeutic protein from the immobilized cells, while also causing a metabolic obstacle to implantation by decreasing diffusion of oxygen, which then leads to graft failure.

1.2. Alginate Gels: Elaboration of Beads and Capsules

Under controlled conditions, alginates will form gels with a large number of divalent cations. The rigidity of the ionic alginate gels increases generally with the affinity of the ion in the order: Mn>Co>Zn>Cd>Ni>Cu>Pb>Ca>Sr>Ba (17). However, most of these ions cannot be used for immobilization of therapeutic active cells. In general, Ca+2 is the most frequently employed ion for such purposes because of its low toxicity. The gel-forming ability of the alginate is mainly related to its content of guluronic acid. In fact, diaxially linked G blocks create a cavity that acts as a binding site for the cation (Fig. 3).

M-fractions

M-fractions

^^ Cross-linked G-blocks

^^ Cross-linked G-blocks

Enzyme Immobilisatiobn

M-rich alginates

G-rich alginates

Fig. 3. Network structure in gels made from alginates with guluronic blocks of different lengths.

M-rich alginates

G-rich alginates

Fig. 3. Network structure in gels made from alginates with guluronic blocks of different lengths.

Gel strength depends on the molecular size and the composition of the alginates. For example, high-G alginates bind the divalent ions better than alginates with low content in guluronic blocks, making gels with in general highest mechanical strength (18), more stability to chelating compounds (19), and higher porosity. The latter can be explained because high-G alginates, with their long G-blocks and short elastic segments, create more open and static networks compared with low-G alginates. Other variables that must be taken into account when preparing alginate beads include: type and viscosity of the alginate, pH, temperature, presence of sequestrants in the cross-linking solution (such as EDTA or citrate), source, and concentration of calcium ions, and microencapsulation method.

One alternative for making more stable and less porous gels is to fabricate homogeneous core networks. Because alginate gels are usually formed by diffusing cross-linking ions into an alginate solution, gels with varying degrees of anisotropy with respect to polymer concentration can be formed by controlling the kinetics of the gel formation (20). Simply by addition of nongelling ions (such as sodium or potassium) to the cross-linking solution, the coupled diffusion between alginate and counter ions is impaired and more homogeneous gels are formed. Another way to improve the stability and reduce the porosity of alginate beads was proposed 25 yr ago by Lim and Sun and it consisted of coating the cell-loaded cross-linked beads with a polycation such as poly-L-lysine (PLL) (21). By simply suspending the the negatively charged alginate droplets in the positively charged PLL solution, a detectable and mechanically resistant semipermeable membrane will be formed around the beads in 5-10 min. In a subsequent step, capsules are coated again with an alginate layer to improve the biocompatibility of the system. This system will be explained in detail later. The pioneering work of Lim and Sun opened the door to innumerable novel cell encapsulation protocols in which the elaboration conditions, the cells, the polycations, or all of them were changed to

Alginate Immobilized Cell

Fig. 4. Elaboration of cell-loaded alginate-PLL-alginate microcapsules using an electrostatic droplet generator. Initially, cells and alginate solution are mixed and the suspension is extruded using the electrostatic device. Because only CaCl+2 is used as cross-linking solution, inhomogeneous core beads with improved mechanical stability will be formed. The addition of NaCl to the collecting solution will result in homogeneous core beads (1). In a second step, beads are washed and coated with a 0.05% (w/v) PLL solution for 5 min (2) and finally the resulting capsules are incubated with a 0.1% (w/v) alginate solution to create the outer biocompatible layer (3).

Fig. 4. Elaboration of cell-loaded alginate-PLL-alginate microcapsules using an electrostatic droplet generator. Initially, cells and alginate solution are mixed and the suspension is extruded using the electrostatic device. Because only CaCl+2 is used as cross-linking solution, inhomogeneous core beads with improved mechanical stability will be formed. The addition of NaCl to the collecting solution will result in homogeneous core beads (1). In a second step, beads are washed and coated with a 0.05% (w/v) PLL solution for 5 min (2) and finally the resulting capsules are incubated with a 0.1% (w/v) alginate solution to create the outer biocompatible layer (3).

improve some key properties such as permeability, biosecurity, reproducibility, biocompatibility, and mechanical stability (22-25).

Another focus of interest is the selection of an appropriate microencapsulation method. In general, elaboration of cell-loaded alginate-PLL-alginate capsules involves two steps: droplet formation and gelation of the droplets in a cross-linking solution. The former can be realized using different techniques, such as the application of a coaxial air jet or a liquid flow to increase the force acting on nascent drops, the use of an electrostatic potential between the capillary and the collecting solution to improve drop formation while reducing size distribution, or the employment of a vibrating or a rotating capillary jet breakage to break the alginate-cell suspension jet into small droplets.

In this chapter, we will focus on the immobilization of cells in alginate gels to create alginate-PLL-alginate microcapsules using an electrostatic droplet generator. The encapsulation system (Fig. 4) is basically composed of a droplet generator device and a peristaltic pump governaed by a number of variables that need to be optimized for optimal microcapsule elaboration. The effect of such variables on the size of the resulting beads will be discussed later.

1.3. Immobilization of C2C12 Cells in Alginate-PLL-Alginate Microcapsules

C2C12 myoblasts have been selected as model cells for their immobilization because they have been broadly employed in literature as somatic gene therapy approach using cell encapsulation technology. Furthermore, C2C12 cells can differentiate terminally into myotubes both in vitro (26) and after immobilization (27). In this way, they could reach a numerically stable population set by the carrying capacity of the microcapsule, which is an advantage to redefine the dosage for in vivo application. On the other hand, the microcapsules proposed here are made of a calcium-alginate matrix and PLL-alginate semipermeable membrane.

2. Materials

2.1. General Reagents

1. Sodium alginate (FMC Biopolymer, Norway or Sigma, St. Louis, MO; see Note 1).

2. Poly-L-lysine (PLL) hydrochloride (mol wt: 15,000-30,000; Sigma).

3. Calcium chloride solution, mannitol, sodium chloride (Sigma).

4. Hanks solution (see Note 2).

2.2. Cell and Culture Mediums

1. C2C12 myoblast cells (from LGC/ATCC batch number CRL-1772).

2. Culture mediums: DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution for the myoblast cells. To differentiate the myoblast into myotubes the complete medium was supplemented with 2% horse serum instead of FBS. Reagents from Gibco-BRL (Invitrogen S.A., Spain).

3. Cell passes every 2 d. Cell cultured at 37°C in a humidified 5% CO2/95% air atmosphere.

4. The flasks, pipets, centrifuge tubes, and the remaining materials must be sterile.

2.3. Electrostatic Droplet Generator

The following parameters must be optimized in order to fabricate uniform and reproducible microcapsules using an electrostatic device: needle diameter, electrostatic voltage, flow-rate, and distance between the needle and the gelling solution. The influence of each parameter on the size of the droplets is reviewed in Table 1. The size of the capsule is critical because it has direct effect on mass transfer of nutrients and especially of oxygen, a key parameter for cell survival. Some studies show that anoxia can occur in the center of spheres of 250 ^m in diameter (28). Smaller microcapsules have been shown to present better biocompatibility (29,30) than larger ones but, on the contrary, they show lower stability (31). Experts in the field have considered 400-450 ^m as an optimal diameter for cell immobilization. Using an electrostatic device it is possible to obtain capsules of 250 ^m in diameter with aceptable size dispersions.

3. Methods

3.1. Preparation of the Cell Pellet (see Note 3)

1. Wash the cells attached to flasks several times with PBS solution, pH 7.4.

Table 1

Effect of the Main parameters Affecting Microcapsules Production by Electrostatic Droplet Generator (unpublished data)

T Needle diameter T Diameter of the droplets

T Electrostatic voltage i Diameter of the droplets

T Flow rate No significant changes

T Distance from needle to solution T Diameter of the droplets

Counting Grid Haemocytometer Journals

Fig. 5. The hemocytometer is a simple method for the enumeration of eukaryotic and prokaryotic cells. It is made of a glass slide that is viewed under a light microscope that consists of two identical counting chambers with a series of grids and a fragile (expensive) coverslip. Under the microscope it is possible to see nine fields that are each 1 mm square. However, only cells deposited in the four peripheric fields will be counted (right). On the left, an extended vision of one of the field in which the the circles represent cells suspended in the chamber between the slide and the cover slip.

Fig. 5. The hemocytometer is a simple method for the enumeration of eukaryotic and prokaryotic cells. It is made of a glass slide that is viewed under a light microscope that consists of two identical counting chambers with a series of grids and a fragile (expensive) coverslip. Under the microscope it is possible to see nine fields that are each 1 mm square. However, only cells deposited in the four peripheric fields will be counted (right). On the left, an extended vision of one of the field in which the the circles represent cells suspended in the chamber between the slide and the cover slip.

2. After washing, add between 5 and 10 mL of trypsin-ethylene diamine tetraacetic acid (EDTA) (Gibco-BRL) to the flasks to provoke the detachment of the cells.

3. Once cells are detached, add 3 mL of complete culture medium to the flasks to stop the effect of the trypsin-EDTA solution.

4. At this point, transfer the cell suspension to a 50 mL centrifuge tube and determine the cell density using an hemocytometer as shown in Fig. 5.

5. Centrifuge the cell suspension to obtain a pellet.

6. Redisperse the cell pellet in a known volume of alginate to obtain the desired final cell density.

3.2. Preparation of the Reagents (see Note 4)

1. Prepare a 1.5% (w/v) sodium alginate solution (LVG: low viscosity alginate rich in gulutonic blocks) in Milli-Q water with 1% (w/v) mannitol. Ten milliliters of the alginate solution is enough for three batches of 2-mL of capsules. This solution will be extruded to form the alginate droplets

2. Prepare a 0.05% (w/v) PLL solution in Milli-Q water with 0.9% (w/v) NaCl. Twenty milliliters of this solution are needed to coat 2 mL of alginate droplets.

3. Prepare a 0.1% (w/v) sodium alginate solution in Milli-Q water with 1% (w/v) mannitol. Twenty milliliters of this solution are needed to create a outer layer in 2 mL of alginate droplets.

4. Prepare a 55-mM calcium chloride solution in Milli-Q water with 1% (w/v) mannitol. Eighty milliliters of this solution are needed per capsule batch.

5. Sterilize all the solutions with a 0.22-|im filtration unit.

3.3. Elaboration of Cell-Loaded Microcapsules (see Note 5)

1. Mix the cell pellet with the corresponding volume of alginate solution to obtain the desired cell density.

2. Transfer the alginate-cell suspension to a syringe and connect the latter to the peristaltic pump.

3. Connect the syringe to a 0.4-mm needle using a 22-cm-length plastic tube. In this way, the alginate-cell suspension will be transferred from the peristaltic pump to the electrostatic generator

4. Incorporate the cross-linking solution inside the electrostatic device (see Note 6).

5. Switch on the peristaltic pump and the electrostatic device. Variables: needle diameter 0.35 mm, voltage 8 kV, flow-rate 5.9 mL/h, and distance from needle to gelling solution 25 mm (see Note 7). These variables enable the elaboration of microcapsules with a mean diameter of 450 |m

6. Extrude the alginate-cell suspension until the desired bead number is obtained.

7. Stop the electrostatic droplet generator and the peristaltic pump. Be aware that there is no alginate-cell suspension in the needle because without electrostatic potential big droplets will be formed.

8. Leave the cell-loaded alginate beads in the calcium chloride solution for 5-10 min to induce the total polymerization of the alginate.

9. Filter the bead solution to eliminate the satellite peaks (see Note 8).

10. Wash the filtered beads with Hanks' solution.

11. Tranfer the beads to a 50-mL centrifuge tube and incubate them with a 0.05% (w/v) PLL solution for 5 min (see Note 9).

12. Filter the cell-loaded capsules to eliminate the nonbound PLL.

13. Wash the capsules again with Hanks' solution.

14. Tranfer the beads to another 50-mL centrifuge tube and incubate them with a 0.1% (w/v) alginate solution for 5 min.

15. Wash and filter the capsules and culture them in complete culture medium.

16. Additionally, it is possible to prepare liquefied capsules by incubating the solid spheres in citrate solution (55 mM for 5 min) as shown in Fig. 6 (32-34).

4. Notes

1. Selection of alginates must take into account some variables, such as viscosity and monomeric acid composition. FMC Biopolymer, Norway, sells four differ-

Raft Systems Incorporate Alginate Gels

Fig 6. The treatment of solid core capsules with citrate provokes the de-gelling of the capsule by the exchange of calcium ions by sodium ions. This induces the entrance of water inside the capsule increasing the osmotic pressure. As a consequence an increase in the diameter of the capsule and the free movement of the cells within the matrix is obtained.

Fig 6. The treatment of solid core capsules with citrate provokes the de-gelling of the capsule by the exchange of calcium ions by sodium ions. This induces the entrance of water inside the capsule increasing the osmotic pressure. As a consequence an increase in the diameter of the capsule and the free movement of the cells within the matrix is obtained.

ent purified alginates: LVG, low-viscosity guluronic acid rich alginate; LVM, low-viscosity mannuronic acid rich alginate; MVG, medium-viscosity guluronic acid rich alginate; and MVM, medium-viscosity mannuronic acid rich alginate.

2. Hanks' solution, pH 7.4, is composed of 125 mMNaCl , 4.8 mMKCl, 1.3 mM CaCl2 , 25 mM HEPES, 1.2 mM Mg2SO4, 1.2 mMKH2PO4, 1 mM ascorbic acid, and 5.6 mM glucose.

3. Cells selected for immobilization need to be detached from the flasks and counted using a hemocytometer (Sigma; see Fig. 5).

4. Cell microencapsulation is a technological process that must be carried out in sterile conditions to avoid the risk of any contaminations of the immobilized cells.

5. It is convenient to adjust the pH of the solutions to 7.4

6. Nonhomogeneous microcapsules are prepared using calcium chloride as cross-linking solution while homogeneous ones need the addition of sodium chloride.

7. Check that the extrusion process is correct and that suitable and uniform droplets are being fabricated.

8. Satellites are formed by breakage of the fine filament between the droplet and the needle tip just before separation, resulting in secondary peaks.

9. The concentration and incubation time of the polycation is optional. The increase of both the concentration and the coating time will provoke a reduction in the size of the capsules and a thicker membrane with a reduced permeability properties.

References

1. Vacanti, J. P. and Langer, R. (1999) Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation.

Lancet 354, 32-34.

2. Thomas, A., Harding, K. G., and Moore, K. (2000) Alginates from wound dressings activate human macrophages to secrete tumour necrosis factor-a. Biomaterials 21, 1797-1802.

3. Okada, H. (1997) One and three month release injectable microspheres of the LH-RH superagonist leuprorelin acetate. Adv. Drug Deliv. Rev. 28, 43-70.

4. Cleland, J. L. (1997) Recombinant human growth hormone poly(lactic-co-glycolic acid) microsphere formulation development. Adv. Drug Deliv. Rev. 28, 71-84.

5. Yang, H. and Wright J. R. (1999) Calcium alginate. In: Cell Encapsulation Technology and Therapeutics, (Kühtreiber, W. M., Lanza, R. P., and Chick, W. L. eds.) Birkhäuser, Boston, MA, pp. 79-89.

6. Bowersock, T. L., Hogenesch, H, Suckow, M., et al. (1996) Oral vaccination with alginate microsphere systems. J. Control. Rel. 39, 209-220.

7. Prisant, L. M., Bottini, B., DiPiro, J. T., and Carr, A. A. (1992) Novel drug-delivery systems for hypertension. Am. J. Med. 93, 459-559.

8. Zhou, S., Deng, X., and Li, X. (2001) Investigation on a novel core-coated microspheres protein delivery system. J. Control. Rel. 75, 27-36.

9. Orive, G., Hernández, R. M., Gascón, A. R., et al. (2003) Cell encapsulation: promise and progress. Nat. Med. 9, 104-107.

10. Krefting, A. (1896) An improved method of treating seaweed to obtain valuable products therefrom. British Patent no. 11538.

11. Haug, A., Larsen, B., and Smidsrad, O. (1966) A study of the constitution of alginic acid by partial acid hydrolisis. Acta Chem. Scand. 20, 183.

12. Smidsrød, O. and Skjäk-Brak, G. (1990) Alginate as immobilization matrix for cells. Trends Biotechnol. 8, 71-78.

13. Espevik, T., Otterlei, M., Skjäk-Brak, G., Ryan, L., Wright, S. D., and Sundan, A. (1993) The involvement of CD14 in stimulation of cytokine production by uronic acid polymers. Eur. J. Immunol. 23, 255-261.

14. Soon-Shiong, P., Otterlei, M., Skják-Brsk, G., et al. (1991) An immunology basis for the fibrotic reaction to implanted microcapsules. Transplant. Proc. 23, 758-759.

15. Clayton, H. A., London, N. J., Colloby, P. S., Bell, P. R., and James, R. F. (1991) The effect of capsule composition on the biocompatibility of alginate-poly-L-lysine capsules. J. Microencapsul. 8, 221-233.

16. Cole, D. R., Waterfall, M., McIntyre, M., and Baird, J. D. (1992) Microencapsulated islet grafts in the BB/E rat: a possible role for cytokines in graft failure. Diabetologia 35, 231-237.

17. Smidsrad, O. (1974) Molecular basis for some physical properties of alginates in the gel state. J. Chem. Soc. Faraday. Transact. 57, 263-274.

18. Martinsen, A., Skjäk-Brak, G., and Smidsrad, O. (1989) Alginate as immobilization material: I. Correlation between chemical and physical properties of alginate gel beads. Biotechnol. Bioeng. 33, 79-89.

19. Skjäk-Brak, G. and Martinsen, A. (1991) Applications of some algal polysaccharides in biotechnology. In: Seaweed Resources in Europe: Uses and Potential

(Guiry, M. D. and Blunden, G., eds.) John Wiley & Sons Ltd., New York, NY, pp. 219-257.

20. Thu, B., Gäserad, O., Paus, D., et al. (2000) Inhomogeneous alginate gel spheres: An assesment of the polymer gradients by synchrotron radiation-induced X-ray emmision, magnetic resonance microimaging, and mathematic modeling. Biopolymers 53, 60-71.

21. Lim, F. and Sun, A. M. (1980) Microencapsulated islets as a bioartificial endocrine pancreas. Science 210, 908-910.

22. Orive, G., Hernández, R. M., Gascón, A. R., et al. (2004) History, challenges and promises of cell microencapsulation. Trends Biotechnol. 22, 87-92.

23. Bartkowiak, A., Canaple, L., Ceausoglu, I., et al. (1999) New multicomponent capsules for immunoisolation. Ann. NY Acad. Sci. 18, 134-145.

24. Chia, S. M., Wan, A. C. A., Quek, C. H., et al. (2002) Multi-layered microcapsules for cell encapsulation. Biomaterials 23, 849-856.

25. Dautzenberg, H., Schuldt, U., Grasnick, G., et al. (1999) Development of cellulose sulfate-based polyelectrolyte complex microcapsules for medical applications. Ann. NY Acad. Sci. 18, 46-63.

26. Blau, H. M., Pavlath, G. K., Hardeman, E. C., et al. (1985) Plasticity of the differentiated state. Science 230, 758-761.

27. Hortelano, G., Al-Hendy, A., Ofosu, F. A., and Chang, P. L. (1996) Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B. Blood87, 5095-5103.

28. Colton, C. K. (1995) Implantable biohybrid artificial organs. Cell Transplantation 4, 415-436.

29. Robitaille, R., Pariseau, J. F., Leblond, F., Lamoureux, M., Lepage, Y., and Halle, J. P. (1999) Studies on small (<350 pm) alginate-poly-L-lysine microcapsules. III. Biocompatibility of smaller versus standard microcapsules. J. Biomed. Mater. Res. 44, 116-120.

30. Leblond, F. A., Simard, G., Henley, N., Rocheleau, B., Huet, P. M., and Halle, J. P. (1999) Studies on smaller (similar to 315 pM) microcapsules: IV. Feasibility and safety of intrahepatic implantations of small alginate poly-L-lysine microcapsules. Cell Transplant. 8, 327-337.

31. Strand, B. L., Gäserad, O., Kulseng, B., Espevik, T., and Skjäk-Brak, G. (2002) Alginate-polylysine-alginate microcapsules—effect of size reduction on capsule properties. J. Microencap. 19, 615-630.

32. Orive, G., Hernández, R. M., Gascón, A. R., Igartua, M., Rojas, A., and Pedraz, J. L. (2001). Microencapsulation of an anti VE-cadherin antibody secreting 1B5 hybridoma cells. Biotechnol. Bioeng. 76, 285-294.

33. Thu, B., Bruheim P., Espevik, T., Smidsrad, O., Soon-Shiong, P., and Skjäk-Brsk G. (1996) Alginate polycation microcapsules. I. Interaction between alginate and polycation. Biomaterials 17, 1031-1040.

34. Thu, B., Bruheim P., Espevik, T., Smidsrad, O., Soon-Shiong, P., and Skjäk-Brsk G. (1996) Alginate polycation microcapsules. II. Some functional properties. Biomaterials 17, 1069-1079.

Was this article helpful?

+1 0
Diabetes Sustenance

Diabetes Sustenance

Get All The Support And Guidance You Need To Be A Success At Dealing With Diabetes The Healthy Way. This Book Is One Of The Most Valuable Resources In The World When It Comes To Learning How Nutritional Supplements Can Control Sugar Levels.

Get My Free Ebook


Responses

Post a comment