Biopolymer Composition for Encapsulating Cells, Method for Producing a Biopolymer Composition for Encapsulating Cells, Method for Promoting Cell Cytoprotection and Use of a Biopolymer Composition for Encapsulating Cells

The present invention relates to a biopolymer composition for encapsulating cells, containing alginate, at least one glycosaminoglycan compound, preferably chondroitin sulfate, and at least one component of the extracellular matrix, preferably laminin, and to the process for producing the biopolymer composition. Also disclosed are a method for promoting cytoprotection using this composition, and the use of this composition for preparing a medicament useful in cell transplantation.

DESCRIPTION OF THE INVENTION

The combined deficient factors found in the prior art for the encapsulation of cells led to the development of a new biopolymer composition for the encapsulation of cells which not only presents improved mechanical strength but also improves the survival and function of the encapsulated cells, or presents higher biocompatibility and stability compared to other microcapsules presented in the literature. These attributes are achieved with the use of a specific combination of components, in specific amounts that eliminate the need of the use of polycations and avoid undesirable complicated methods of production.

The present invention also relates to a method of producing said composition by specifically adjusted parameters that guarantee the efficiency of the composition in a shorter production time. Still objects of the present invention are a method for promoting the cytoprotection and a use of the developed biopolymer composition for the manufacture of a medicament useful for cell transplantation.

It was found in this new biopolymer composition superior properties and benefits that ensure the biocompatibility (FIG. 7), the function and viability of microencapsulated cells, which is essential not only for therapeutic or prophylactic activities are achieved, but also to ensure that maximum longevity and viability of the transplanted cells.

The researchers of the present invention found that the apoptosis and cellular stress can be reduced by adding elements of the extracellular matrix such as laminin in biopolymers systems based on alginate and chondroitin sulfate, in order to prevent cell death and also promote proliferation and cell viability (FIGS. 2 and 8).

In addition, the same researchers found that addition of chondroitin sulfate and laminin to the composition of the biopolymer alginate ensures narrowing of the pores and protection of microencapsulated cells of molecules and cells of the immune system, solving the problem that the micro-encapsulation with alginate gelled with calcium creates pores to produce microcapsules with larger dimensions. This situation increases the resistance of the microcapsules and improves survival and functionality of the encapsulated cells through cytoprotective molecular signals (FIGS. 2 and 8).

The combination of alginate, glycosaminoglycans components, such as chondroitin sulfate, and extracellular matrix components such as laminin, for the manufacture of microcapsules which are gelled by solution of divalent cations, such as barium ions, was introduced as a great improvement over the current state of the art by adding biological properties advantageous to the microcapsules. Even barium compound is toxic to the body once it establishes ionic bonds within the microcapsule mesh it becomes unavailable to the surroundings and thus no significant toxicity to the individual receiving the implant and the encapsulated cells themselves (FIG. 1).

The current state of the art does not disclose compositions for the encapsulation of biopolymer-based cells in alginate and glycosaminoglycan components of the extracellular matrix components. Furthermore, there are known methods for promoting cytoprotection using such compositions, as well as the use of these particular compositions for the preparation of a medicament useful in cell transplantation.

One of the objects of this invention relates to a biopolymer composition for the encapsulation of cells based on alginate, glycosaminoglycans components, such as chondroitin sulfate, and extracellular matrix components.

In the composition according to the present invention the ratio of alginate:chondroitin sulfate is about 4:1 and laminin is present in a final concentration of about 10 μg·mL−1.

Another object of this invention relates to a method for promoting the protection of encapsulated cells (cytoprotection) by using a biopolymer composition based on alginate, glycosaminoglycans components, such as chondroitin, and extracellular matrix components such as laminin, according with the present invention.

It is also another object of this invention to the use of a composition based on alginate biopolymer, components glycosaminoglycans such as chondroitin and extracellular matrix components such as laminin, according to the present invention for the preparation of a medicament useful in cell transplantation.

There are several advantages of the present invention over the prior art, the same being scored as follows:

1) developed biopolymer composition induces changes in the expression of important genes related to apoptosis. The effector caspase-3 gene, which is activated late in the apoptotic cascade, has its reduced expression in cells microencapsulated biopolymer with the composition of this invention. In addition, the anti-apoptotic bcl-2 gene, whose product is important for protecting cells against apoptosis mechanisms has its increased expression in cells microencapsulated with said composition (FIG. 2). The expression of anti-apoptotic proteins Bcl-xL and XIAP are also elevated in microencapsulated islets with Alg-SC-LN microencapsulated islets compared with Alg-SC (FIG. 8).

2) The claimed composition biopolymer increases the ratio of gene expression of bcl-2 and bax (bcl-2/bax), which shows that microencapsulation with such a composition protects cells against apoptosis. Increased ratios of the expression of bcl-2/bax and bcl-xL/bax at both the gene and protein level, showed a decrease in the susceptibility of the cell to apoptosis (Brown et al., 2007) (FIG. 2).

3) The developed biopolymer composition decreases expression of the genes MCP-1 and hsp70, both related to cellular stress (FIG. 2).

4) The developed composition biopolymer in pancreatic islet cells microencapsulation models increases the expression of the rat insulin 1 gene, which can lead to an increase in the percentage of β cell precursors which differentiate into mature β cells. This composition also restores cell-cell and cell-matrix contact that is essential for maintenance of cell viability and function (FIG. 2).

5) The developed method for cell encapsulation uses a reduced number of pancreatic islet cells required for cell transplantation. For the current state of the art, the mean number of rat islets required to reverse the mice diabetic state is about 1,450. By using the biopolymer composition of the present invention, it was possible to maintain normoglycemia in diabetic mice over 200 days via an implant of only 750 microencapsulated islets, which is 48% less than the amount islets reported in the art (FIG. 3). The longevity of the graft was significantly higher in mice that received microencapsulated islets with Alg-SC-LN (FIG. 6). The quality of the islets was assessed by transplantation throughout the test oral glucose tolerance test (OGTT) (FIGS. 4 and 5). This difference is significant when considering that this reduction can lead to a pancreas saving for islet transplantation in humans, since the average surgeries require islets (encapsulated or not) extracted from two or more human pancreas in order to the diabetic patient receiver has normalized blood glucose.

6) In pancreatic islet cell microencapsulation model, the developed biopolymer composition of the present invention gets normoglycemia after transplantation in a shorter period of time compared to the current level of technology. The presence of extracellular matrix components such as laminin in the composition of this invention reduces by up to 4 days to reach normoglycemia after transplantation. In the clinic, this can mean fewer days of hospitalization due to a faster recovery of the patient (FIG. 3).

7) The biocompatibility of biomaterials Alg-SC and Alg-SC-LN was demonstrated by testing in which these biomaterials-containing capsules were placed in culture with macrophages. The expression of pro-inflammatory cytokines IL-l-beta and TNF-alpha was not stimulated in the macrophages cultured with these biomaterials in contrast with the increased expression of these cytokines in macrophages cultured with alginate grom Sigma manufacturer or in the presence of LPS (FIG. 7.)

Alternative forms or modifications within the scope of the present invention will become readily detectable by person skilled in the art from reading the following specifications and references.

The method of producing the biopolymer composition consists in the mixture of ideal proportions of alginate with at least one component glycosaminoglycan, preferably chondroitin sulphate, together with at least one extracellular matrix component, preferably laminin.

This mixing is done at the time of microencapsulation, as well as mixing the biomaterial with cells. This final mixture should be performed as quickly as possible because of the time consuming and direct contact with biomaterial ungelated cells can cause harmful effects to the viability and functionality of these. This procedure should be performed only when the entire apparatus of microencapsulation is prepared for making the microcapsules. The cells should be sedimented by centrifugation, and homogenized thoroughly in the biomaterial, and adding this mixture to a syringe, which is connected to the device that produces the microcapsules.

The cell encapsulation may be directed to stem cells, muscle cells, pancreatic cells, chondrocytes, liver cells, cells of the central nervous system, renal cortex cells, vascular endothelial cells, skin cells, parathyroid and thyroid cells, adrenal cells, cells thymic cells, ovarian germline cells, embryos or cells which include recombinant genetic material.

For making the microcapsules, it uses a syringe pump to expel the mixture of the biopolymer with the cells. By applying an air flow around the coaxial needle, it is possible to detach the droplet at the desired time, and therefore control the size of the microcapsules. The distance between the needle tip and the gelling solution is adjusted to microcapsules delicately reach the bottom of the container where they are deposited, thus avoiding mechanical shock which can cause deformations. The height between the needle tip and exits the biomaterial containing the cells and the gelling solution may be between 5 and 10 cm. The flow of biomaterial containing the cells can be expelled through the needle at a flow ranging between 15 and 30 mL·h−1. The coaxial air flow can vary between 2.0 and 2.5 L·min−1, which can generate microcapsules still considered optimum between 500 and 1000 μm.

The diameter of the microcapsules is dependent on the ion used for the gel formation, on the gel solution concentration and the flow of air. After detachment of the needle, the microcapsules fall into a solution of polymerization (gel formation) comprising divalent ions, such as BaCl2or CaCl2, preferably BaCl2, and it is buffered with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid—HEPES at pH 7.4.

It is important to note that gel formation is conducted under physiological conditions, causing no harm to the cells. When it comes into contact with the gelling solution, the biomaterial passes from the soluble state to gel state. Laminin and chondroitin sulfate, which are contained in the biomaterial, remain anchored in the alginate-ion networks, helping to close the formed mesh and forming pores of suitable size.

At the end of the process, the microcapsules remain in the solution for 5 minutes. After this incubation step, the microcapsules are rapidly filtered. The excess ion used for the gelation of the biomaterial is removed by successive washing of the microcapsules in 0.15 M NaCl.

The microcapsule formed around the cells is permeable to insulin, glucose, nutrients and oxygen and impermeable to molecules and cells of the immune system, preventing direct contact between the transplanted graft and the patient's immune system in case of cell transplantation or cell therapy.

Other application techniques can also be attributed to the invention as the large-scale production of molecules derived from cells, reproductive technology and cell culture-dependent contact with other cells and/or proteins, and others, such as the food industry (microencapsulation of yeast for brewing beer and microencapsulation of seeds) and in the pharmaceutical industry (microencapsulation of biopharmaceuticals or chemotherapy).

The following examples are provided in order to illustrate the main aspects of the present invention. It should be noted that for those who know the state of the art, the descriptions below, used by the inventors of this invention may be regarded as one of the various ways in which the invention can be achieved. It is understood that changes invention can be used in effect, obtaining also results equal or similar to those described in the scope of this invention. The invention is further explained by the following examples.

EXAMPLES

Preparation of the Biomaterial of the Biomaterial and Mixed with the Cells Whether to Microencapsulate

The biopolymer is diluted in NaCl 0.15 mol L−1to a final concentration of 1.2% alginate. The alginate biopolymer is formed by alginate:chondroitin sulfate in a ratio of 4:1 and laminin-1 is added in this mixture to a final concentration of 10 μg·mL−1. The cell suspension should be carefully and fast homogenized in the solution of biopolymer-NaCl. The gelling solution is 0.02 mol·L−1Barium Chloride plus 20 mmol·L−1HEPES (Sigma), pH 7.2.

Preparation of Microcapsules and Parameters Used to Obtain a Microcapsule with Good Size, Shape and Stability

10,000 islets are used per mL alginate, laminin, chondroitin sulfate or 1.5×106cells·mL−1biopolymer. The capsules were obtained by extruding the solution containing the biomaterial islets (or cells) by a microneedle at a flow rate of 19.9 mL·h−1controlled by a syringe pump (SP 500 JMS do Brasil, Campinas, Brazil). By applying 2.2 L·min−1air flow (air medicinal, Air Products Brasil Ltda.) around the needle. After detachment of the needle drop, the microcapsules falls into a polymerization solution (gelling) comprising BaCl2. With the above determined flow it is obtained microcapsules with a diameter of about 700-800 μm.

The distance between the needle tip and the gelling solution was adjusted to 7.5 cm. At the end of the process, the microcapsules remain in the solution for 5 minutes. After this incubation step, the microcapsules are rapidly filtered and washed with 0.15 mol·L−1NaCl.