Technology of mammalian cell encapsulation

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Abstract

Entrapment of mammalian cells in physical membranes has been practiced since the early 1950s when it was originally introduced as a basic research tool. The method has since been developed based on the promise of its therapeutic usefulness in tissue transplantation. Encapsulation physically isolates a cell mass from an outside environment and aims to maintain normal cellular physiology within a desired permeability barrier. Numerous encapsulation techniques have been developed over the years. These techniques are generally classified as microencapsulation (involving small spherical vehicles and conformally coated tissues) and macroencapsulation (involving larger flat-sheet and hollow-fiber membranes). This review is intended to summarize techniques of cell encapsulation as well as methods for evaluating the performance of encapsulated cells. The techniques reviewed include microencapsulation with polyelectrolyte complexation emphasizing alginate–polylysine capsules, thermoreversible gelation with agarose as a prototype system, interfacial precipitation and interfacial polymerization, as well as the technology of flat sheet and hollow fiber-based macroencapsulation. Four aspects of encapsulated cells that are critical for the success of the technology, namely the capsule permeability, mechanical properties, immune protection and biocompatibility, have been singled out and methods to evaluate these properties were summarized. Finally, speculations regarding future directions of cell encapsulation research and device development are included from the authors’ perspective.

Introduction

Cell encapsulation aims to entrap viable cells within the confines of semi-permeable membranes. The scientific literature suggests that membranes are expected to be permeable for transport of molecules essential for cell survival, but not to allow the transport of molecules larger than a desired critical size. Despite its nonphysiological nature, the permselective capsule environment has been shown to support cellular metabolism, proliferation, differentiation and cellular morphogenesis. The primary impetus behind the recent development of cell encapsulation technologies has been the desire to transplant cells across an immunological barrier without the use of immunosuppressant drugs. The latter, due to their systemic administration, are associated with unwanted side effects due to non-specific suppression of the immune system that leads to a variety of undesired complications (e.g., opportunistic infections, failure of tumor surveillance, as well as adverse effects on the encapsulated tissues) [1], [2], [3]. These complications cannot justify the ethical use of systemic immunosuppressants for a significant fraction of patients who are candidates for cell transplants and provide the impetus to develop alternative methods to overcome the immune rejection process. By surrounding a transplant with a membrane barrier, the access of a host’s immune system to the transplant can be physically prevented. The feasibility of transplanting cells in immunoprotective membranes is under study for the treatment of a wide variety of endocrine such as anemia [4], dwarfism [5], hemophilia B [6], kidney [7] and liver failure [8], pituitary [9] and central nervous system (CNS) insufficiencies [10], and diabetes mellitus [11] (see other chapters in this Volume). Besides transplantation, encapsulated cells are being pursued for a variety of other applications (Table 1). The common thread for all such applications is the need to isolate a desired cell population from an outside environment. Depending on the choice of membrane material and whether membranes are pre-fabricated or fabricated around viable cells, cells will be subjected to different entrapment conditions. The final capsule properties will be variable to suit the needs of a particular application and it is essential, for any encapsulation technology, to tailor capsule properties in a reproducible fashion by the process variables.

An optimal balance has to be maintained among various capsule properties to support cell survival in the context of an expected performance criterion. The mass transport properties of an encapsulation membrane are critical since the influx rate of molecules essential for cell survival and the outflow rate of metabolic end products will ultimately determine the extent of entrapped cell viability (Fig. 1). The metabolic requirements of various cell types are different and, hence, optimal membrane permeability is expected to depend on the choice of cells. Although the role of permeability for particular elements essential for cell survival has been explored (for example, oxygen [27]), no systematic approach has been taken to determine the permeability requirements of particular cell types. An empirical approach has been typically taken to tailor capsule permeability for cell survival. The upper limit of capsule permeability, i.e., molecular mass cut-off (MWCO), will be application dependent. In the case of transplantation, the MWCO is expected to be different whether xenogeneic or allogeneic tissues are destined for engraftment (see Section 5.3 for more details). For bioreactor cultures, the MWCO will vary whether it is desirable to have the cell-derived biomolecules to permeate through the capsule wall. Besides permeability, an important consideration is the availability of an extracellular matrix (ECM) to encapsulated cells [28], [29], especially for anchorage-dependent cells. The ECM not only allows the cells to express their differentiated functions but also helps to organize the cell mass within the capsules for optimal viability [28], [29]. Capsule biocompatibility is critical when encapsulated cells are intended for transplantation since it is the compatibility of a biomaterial with the host that ultimately determines the nature of host response and graft survival. The physicochemical nature of a biomaterial is important to enhance the capsule biocompatibility and exploration of different materials for this purpose has necessitated development of different encapsulation procedures to accommodate the varying physicochemical properties.

This chapter is intended to provide a summary of current cell encapsulation techniques. Since other chapters of this Volume are for specific applications, it was our intention not to emphasize application-specific issues but rather to review the advantages and disadvantages of various cell entrapment processes. A major distinction is in the utilization of ‘macrocapsules’ vs. ‘microcapsules’. In macrocapsules, large groups of cells are enveloped in tube or disc shaped hollow devices while in microcapsules a smaller cell mass is individually entrapped in their own spherical capsule. This manuscript will review techniques of microencapsulation, where cells are entrapped in spherical capsules and beads, or conformally coated. This will be followed by a review of macroencapsulation technology and issues specifically pertinent to macrocapsules. The success of any encapsulation technique will ultimately rely on a systematic evaluation of capsule properties and encapsulated cell performance. Accordingly, various techniques utilized for assessment of capsule properties will be summarized. Where indicated, relationship between the capsule properties and encapsulated cell performance are presented, with a critical emphasis on capsule engineering efforts. Numerous reviews were published over the years on encapsulation technology [30], [31], [32], [33], [34], [35], and the reader is referred to these reviews for different perspectives.

Section snippets

Microencapsulation

Capsules in the 0.3–1.5 mm range have been traditionally refereed to as microcapsules in the cell encapsulation field. Their relatively small size (i.e., large surface area to volume ratio) is considered advantageous from a mass transport perspective. Microcapsules are typically more durable than macrocapsules and difficult to mechanically disrupt. Numerous microencapsulation techniques, fundamentally different in the nature of entrapment mechanism, were developed in independent labs. Besides

Macroencapsulation

Macrocapsules are generally much larger devices compared to microcapsules and typically possess a planar or cylindrical geometry, and a smaller surface-to-volume ratio. Living cells are physically isolated from directly interacting with host tissue by enclosure between two or more selectively permeable flat sheet membranes or within the lumen of a semipermeable hollow fiber. Like their smaller counterparts, these devices rely on the host animal’s own homeostatic mechanisms for the control of

Other encapsulation techniques

A variety of entrapment techniques have been developed to overcome various shortcomings of the micro- and macroencapsulation (Table 2). A silica sol–gel technique has been successfully used to entrap islets in ceramic membranes [192]. This is the first report on the encapsulation of mammalian cells in ceramic membranes. Although detailed investigation of encapsulated cell performance is yet to be performed, this is an intriguing choice of material and it may offer distinct advantages over

Assessment of capsule properties

A critical requirement for the development of a successful cell encapsulation technology is the close relationship between capsule properties and performance of encapsulated cells. The desired capsule properties will obviously depend on a particular application. Even though an expected performance criteria is satisfied (e.g., restoration of normoglycemia in a diabetic model), it is essential to probe the capsule properties that contribute to successful performance. This will facilitate a more

Future considerations

Since the early pioneering period, the technology of mammalian cell encapsulation has developed significantly. We reviewed several approaches to achieve cell encapsulation and possibly to enhance the efficiency and long-term utility of this technology. During recent years, there has been a large increase in the published studies using mammalian encapsulation methods as academic and industrial laboratories drive toward clinical implementation of the technology. In the field of

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