Title:  Chitosan core matrix containing cells encapsulated in a thermoplastic semipermeable membrane

United States Patent:  6,140,089

Assignee:  Brown University Research Foundation (Providence, RI)

Filed:  February 16, 1999

Encapsulated viable cells for implanting are prepared having cells dispersed in a particulate, essentially non cross-linked chitosan core matrix that is enclosed within a semipermeable membrane. The cells are entrapped between chitosan particles of the core matrix and there is essentially no interfacial cross-linking between the core matrix and the membrane. The core matrix provides a physical support for the cells such that the cells are evenly dispersed throughout the core matrix so as to allow their maintenance, growth, proliferation and differentiation. The encapsulated cells may be prepared by mixing viable cells with a solution of chitosan, encapsulating the resultant mixture in a thermoplastic semipermeable membrane, and causing the chitosan to precipitate such as by changing the pH to form the core matrix. Alternatively, the chitosan in solution is precipitated to form the core matrix containing cells, and the core matrix is encapsulated in a semipermeable membrane. Cells encapsulated include neurosecretory cell lines, .beta.-cell-derived cells lines, fibroblasts, myocytes and glial cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that, chitosan may be formed into a three-dimensional, particulate matrix for incorporation into cell encapsulation devices. The chitosan core matrix of the present invention results from the use of a discrete chitosan particle without substantial cross-linking or other chemical attachments between particles. While the precipitate of the present invention is formed substantially in the absence of cross-linking agents, and is essentially uncross-linked, it should be born in mind that there are likely to be free charges on the precipitate surface which remain available for cross-linking under the appropriate conditions. The terms "without substantial cross-linking" or "essentially non cross-linked", etc. for purposes of this invention refer to the total assemblage of the chitosan within an encapsulation device, such that the chitosan is particulate in nature and the individual polymer chains are not covalently connected or ionically cross-linked into a single or a few gel-like structures.

The chitosan core matrices of the present invention entrap cells between particles but not within a continuous three-dimensional matrix as within a cross-linked gel structure (e.g. alginate in the presence of ionic calcium). Such particulate chitosan matrices may be produced by pH dependent precipitation of soluble chitosan substantially in the absence of cross-linking agents. The particulate chitosan matrices may also be produced by placing chitosan-containing capsules in any substantially non-toxic solution having a sufficient ionic content to mask enough of the charges on the chitosan to cause precipitation. Suitable solutions are biological buffers that optionally contain monovalent anions to speed up the rate of precipitation.

The term "cross-linked chitosans" for the purposes of this invention refers to solids or gels that depend upon ionic interactions between polymer chains to retain their solid or gel-like character. Cells in such gels are embedded within a more or less continuous three-dimensional matrix formed by the interconnection of chitosan chains. Cross-linked chitosans are generally formed in the presence of cross-linking agents.

Interfacial cross-links are not the subject of the current invention, and while their formation is not prescribed, their existence generally will not affect the operation of the invention. In other embodiments, minute particles of cross-linked chitosan with essentially no cross-links occurring between particles may be used to form the particulate chitosan matrix. Such particles are formed by the dehydration of cross-linked chitosan gels, and subsequent pulverization (e.g. with mortar and pestle) of the resultant structure to particles less than 100 .mu.m in diameter, preferably less than 50 .mu.m and most preferably less than the size of the cells to be entrapped by them.

In embodiments involving production of particulate chitosan by precipitation, the chitosan precipitation is preferably achieved by placing the chitosan-containing capsules in a buffered solution containing one or more substantially non-toxic monovalent ions such as phosphate, chloride, bicarbonate, iodide or bisulfate. The amount of ions needed for precipitation to occur can be very easily determined by one of ordinary skill in the art, for example, by a titration experiment. Precipitation can also occur by adjusting the pH of the chitosan solution. Virtually any method which removes or masks the charge of a sufficient number of the free amino groups of the chitosan should be suitable. Such methods can involve the reaction of the amino groups with organic substances. Alternately, biocompatible non-chitosan reactive water soluble polymers may be used to induce precipitation. The precipitate provides cell separation and a charged surface for cell interaction. The core matrix is then encapsulated within a membrane or jacket that, upon culture or implantation in an individual, will allow diffusion of nutrients, waste materials, and secreted products, but which is preferably immunoisolatory and blocks the cellular and molecular effectors of immunological rejection. Preferably there is no type of chemical or physical interlinking or bonding between the core matrix and the jacket.

As defined herein, the term "individual" refers to a human or animal subject. The term "tissue" as defined herein refers to cells, cell aggregates, tissue or tissue fragments from either animals or humans.

Chitosan is available from a number of manufacturers and may vary in its purity and % deacetylation both between lots and between manufacturers. Despite the slight differences in solubility properties, many of these chitosans will be useful in the instant invention. However it should be born in mind that the pH-dependent solubility characteristics should be confirmed for any given lot of chitosan. For example, the solubility characteristics of Fluka chitosan flakes in 6 mM HEPES and Protan Seacure Cl have been compared. The Fluka chitosan precipitates at pH>6.3 whereas the Protan chitosan precipitates near pH 6.8.

In one embodiment, the chitosan matrix-forming material is prepared as a soluble solution, mixed with cell-containing media, and then used in a co-extrusion process to form thermoplastic or form-holding encapsulation devices such as fibers or flat sheets. In other embodiments, the chitosan/cell solution is introduced into preformed devices. The matrices of the invention may also be used in the formation of microspheres and for post-production filling of preformed fibers and/or capsules.

The chitosan matrix of the present invention is compatible with several cell types useful in implantable vehicles for the treatment of diseases such as diabetes, Parkinson's disease, and other neurological disorders. In addition, encapsulated myoblasts may be useful as sources of trophic or sprouting factors for supporting peripheral nerve repair or regeneration.

Herein, the term "core matrix" refers to a biocompatible, three-dimensional structure which supports and may enhance cell proliferation and/or cell differentiation.

The chitosan core matrix of the invention is comprised of particulate chitosan which provides or acts as an irregular scaffolding into which cells are free to grow. The matrix provides a large growth area which does not restrict the cells' ability to divide and expand.

Cells which grow well in the matrix of the present invention include CHO cells, fibroblasts, myocytes, neurosecretory cells such as PC12 cells, pancreatic .beta.-cells such as NIT and RIN cell lines, and glial cells such as astrocytes. Cells compatible with the chitosan matrix may be genetically engineered to secrete a desired substance which is heterologous to the compatible cell. For instance, fibroblasts which have been genetically engineered to secrete nerve-growth factor (NGF) are compatible with the present chitosan matrix.

Chitosan is characterized as a poly-n-glucosamine, with a large number of free amino groups. Chitosan is commercially available in a number of forms differing in their number of free amino groups (% deacetylation), degree of purity, molecular weight distribution, and viscosity. In practicing the invention, a preferred type of chitosan has a molecular weight range of 10-1,000 kd preferably 100-300 kd. Lower molecular weight distributions may also be useful.

Preferably, the chitosan has a degree of deacetylation of about 80% to about 90%, preferably 80-85%. A higher degree of deacetylation correlates with a higher number of free amino groups, which are positively charged. Percent deacetylation is important in the regulation of the pH sensitive precipitation of chitosan from the cell/growth media of the current invention. Specifically, chitosans having <50% deacetylation demonstrate solubility over a broader range of pHs (e.g., pH 2 to 11), whereas 80% deacetylated chitosan (Seacure Cl Protan) is soluble at pH 6.3 and precipitates at pH 6.8.

Viscosities for soluble 1% chitosan at pH 4.0 of about 20-80 cp is preferred. The molecular weight distribution of the various uncross-linked chitosan polymer chains within a specific chitosan preparation or lot significantly affects the viscosity of a solution of a given concentration. Additionally, solids (e.g., salts, carbohydrates) or copolymers present in the chitosan solution as well as the actual concentration of the chitosan solution itself, will have significant effects on the viscosity.

Viscosity of the chitosan solution effects the ability to load encapsulation devices, and the speed of precipitation. This has a number of practical consequences for device fabrication and loading. Mechanized capsule fabrication such as the coextrusion process described in U.S. Pat. No. 5,158,881, Aebischer, et al., requires lower viscosity chitosan solutions in the range of 10-150 cps (.about.0.5-2% chitosan), as compared to device fabrication involving manual introduction of the cell/chitosan solution through syringes, etc. where viscosity is a far less critical limitation and can be as great as 1,000 cps (.about.5-10% chitosan).

In one embodiment, to form the core matrix of the invention, the chitosan is first dissolved in an aqueous acid solution, approximate pH 2-pH 4. A variety of acids such as malic acid, citric acid, succinic acid, ascorbic acid, acetic acid or hydrochloric acid may be used to make the aqueous acidic solution. Chitosan is available from several sources (Fluka Chemical Corp., etc.). A preferred source of chitosan is Protan's SeaCure Cl.

Following dissolution of the chitosan in the acidified solution, the pH is raised to a level that is close enough to physiological pH to be tolerated by cells, but still low enough to maintain the solubility of the chitosan. Preferably, the chitosan solution is brought to about pH 6.3-6.5 using a biocompatible buffer such as HEPES, TRIS, or monobasic phosphate. Table 1 summarizes the properties of a number of biologically compatible buffers which may be used in this system. It is advisable to select a relatively weak buffering agent with a useful buffering range that encompasses the pH of precipitation of the chitosan solution. The use of a weak buffer facilitates the adjustment of pH to 7.4 necessary for initiation of chitosan precipitation, as well as the preservation of cell viability (the exposure of cells to pHs other than 7.4 should be minimized). For purposes of this invention, chitosan precipitation occurs preferably in the range of pH 6.5-6.8, the exact value will vary depending upon the degree of acetylation and counter ions present for the particular lot of chitosan used. Therefore, it is always useful to characterize the precise pH conditions for precipitation with a new lot of chitosan. If phosphate buffers are used they should be monobasic. Multi-phosphates should be avoided because groups such as tripolyphosphate lead to undesirable levels of chitosan cross-linking.

Claim 1 of 4 Claims

What is claimed is:

1. Encapsulated viable cells, comprising

viable cells dispersed in a three-dimensional particulate, essentially non-cross-linked, chitosan core matrix encapsulated in a thermoplastic semipermeable membrane,

wherein the chitosan core matrix containing dispersed cells is formed by precipitation of a chitosan solution containing said cells after the solution has been encapsulated in the thermoplastic semipermeable membrane.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.