implantable biohybrid artificial organs

Pergamon

Original Contribution

Cell Transplantation, Vol. 4, No. 4, pp. 415-436, 1995 Copyright @ 1995 Elsevier Science Ltd Printed in the USA. All rights reserved

0963-6897/95 $9.50 + .OO

0963-6897(95)00025-9

IMPLANTABLE BIOHYBRID ARTIFICIAL ORGANS

CLARK K. COLTON

Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 66-452, Cambridge, MA 02139-4307, USA

0 Abstract - Biobybrid artificial organs encompass all devices which substitute for an organ or tissue function and incorporate both synthetic materials and living cells. This review concerns implantable immunoisolation devices in which the tissue is protected from immune rejection by enclosure within a semipermeable membrane. Two critical areas are discussed in detail: (i) Device design and performance as it relates to maintenance of cell viability and function. Atten- tion is focussed on oxygen supply limitation and how it is af- fected by tissue density and the development of materials that induce neovascularization at the host tissue-membrane interface; and (ii) Protection from immune rejection. Our current knowledge of the mechanisms that may be operative in immune rejection in the presence of a semipermeable membrane barrier is limited. Nonetheless, recent studies shed light on the role played by membrane properties in preventing immune rejection, and many studies demonstrate substantial progress towards clinically useful implantable immunoisolation devices.

0 Keywords - Biohybrid artificial organs; Immunoisola- tion; Implantable devices.

INTRODUCTION

The term hybrid artificial organ, referring to a device containing living cells or tissue separated from the body by a synthetic material that substitutes for an organ or tissue function, was first used by Chick and colleagues in 1975 (13) in the context of a new approach for using implanted islets of Langerhans to treat diabetes. Reach et al. in 1981 (53) coined the term bioartificial pancreas to describe the same application. Today, biohybrid artificial organs encompass all devices which incorporate both synthetic materials and living cells. If needed only for acute use, such devices may be applied for treatment of blood by use of an extracorporeal circuit. However, therapeutic application for chronic disease requires de-

vice implantation. To emphasize the original intent of hybrid devices as applied to cell therapies, Scharp and colleagues in 1984 (58) used the term immunoisolation to mean implanted cells or tissues protected from immune rejection by enclosure within a semipermeable membrane.

Biohybrid artificial organs are under study for treatment of a wide variety of diseases, including insulin secretion in diabetes (33,36,63), factor IX in hemo- philia B (11,43), human growth factor in dwarfism (12), erythropoietin in anemia (32), as well as kidney failure (1,16), immunodeficiencies (14), and pituitary problems (28). Recently, the central nervous system has become a major focus, including chronic pain (3 1,56) and neurodegenerative disorders such as Parkinson’s disease (2,3,23,66,69), Alzheimer’s disease (26,70), Hun- tington’s disease, and amyotrophic lateral sclerosis, most of which are being actively pursued by Cytother- apeutics, Inc. Liver assist devices to treat fulminant hepatic failure are under study for acute presurgical clinical management prior to orthotopic liver transplantation or possibly avoidance of transplantation altogether if a sufficient mass of hepatocytes can be re- generated in a reasonable period of time. Treatment of liver disease has been investigated primarily with extracorporal approaches (55), although implantable devices are also being studied (72). The literature on biohybrid artificial organs in several areas has recently been reviewed extensively (4,6,15,17,37,41,46,51). This review will focus on certain technical problems that may limit the clinical application of implantable immunoisolation devices (l&19).

Several features characterize all immunoisolated cell therapy devices: tissue requirements, device configu-

ACCEPTED 3/28/95.

415

416 Cell Transplantation 0 Volume 4, Number 4, 1995

ration, and tissue density. Tissue requirements for immunoisolation devices cover a very large range which is determined by the secretion rate of the desired agent per cell and the amount of active agent required by the body. Because of the very low concentration of neuroactive substances required, central nervous system applications appear to require an order of magnitude of lo6 to lo7 cells, which correspond to a volume of about 1 to 10 ~1. In contrast, implantation of islets of Langerhans for treating diabetes will likely require about lo9 cells corresponding to about 1 ml volume. These parameters are important because the complex- ity and difficulty of the problem increases with the volume of implanted tissue required. Thus, it is not surprising that some central nervous system applications are the first to advance to clinical testing. At the far extreme, replacement of liver function with hepatocytes will require on the order of 10” cells or more than 100 ml volume. This large volume of tissue will make an implantable immunoisolation device approach extremely difficult.

resent the largest cost component for fabrication of the device. Substantial efforts have been made in isolation of primary tissue, especially for pancreatic islets (40), but further improvements are necessary for practical, large-scale processing.

2. Device design and performance. Maintenance of maximum cell viability and function is essential and, in the absence of immune rejection, is limited by the supply of nutrients and oxygen, especially the latter.

3. Protection from immune rejection. This goal applies to any type of implanted tissue, including au- tologous (if made immunogenic in the process of genetic modification), allogeneic, and xenogeneic tissue.

In what follows, the second and third of these issues will be discussed in some detail. Although much of the illustrative material and associated discussion concerns the artificial pancreas for diabetes, virtually all of the concepts are applicable to biohybrid artificial organs in general.

A broad variety of geometric configurations are available for biohybrid devices. An intravascular device usually consists of a tube through which blood flows, on the outside of which is the implanted tissue contained within a housing. The device is implanted as a shunt in the cardiovascular system. Extravascular devices are implanted in tissue in a body space such as the peritoneal cavity. Geometrical alternatives include spherical microcapsules within which tissue is encapsulated, cylindrical tubular membranes containing tissue within the lumen, and planar diffusion chambers comprised of parallel flat sheet membranes between which is placed the implanted tissue.

OXYGEN SUPPLY

Another important characteristic is the tissue density attainable. The high tissue density case, in which all of the available internal space is filled with living, functional cells, is most desirable because it minimizes the size of the implanted device. Until recently, high density could not be achieved because of constraints of device geometry, dimensional size, and supply of oxygen to the encapsulated tissue as discussed in more detail subsequently. Only low tissue density has been successful in maintaining cell viability, invariably with the use an extracellular matrix prepared from a naturally occurring polysaccharide hydrogel such as alginate, agar, or chitosan.

The transport requirements of a biohybrid artificial pancreas are illustrated in Fig. 1. The implanted cells are separated from the body by an immunoisolation membrane which, ideally, prevents components of both the cellular and humoral immune response from entering into the transplanted tissue region but permits passage of the secreted product insulin. At the same time, the transport properties of the membrane and surrounding tissue must permit sufficient assess of nutrients, such as glucose and oxygen, and the removal of secreted metabolic waste products. The islet cells must be supplied with nutrients by diffusion from the nearest blood supply and through surrounding host tissue, the immunoisolation membrane, and the islet tissue itself. This is particularly important for islets because they are normally well perfused by blood and supplied with oxygen at arterial p02 levels. Oxygen gradients will develop in the surrounding host tissue and within the immunoisolation device and may well limit the amount of viable tissue that can be supported in immunoisolation devices. In addition to oxygen, there may be supply limitations for higher molecular weight compounds, for example, the iron-transporting protein transferrin.

There are three critical problem areas in the further The oxygen levels to which the islet cells are exposed development of implantable immunoisolation devices: are important from two standpoints, viability and 1. Supply of tissue. Both primary tissue and cultured function. If the oxygen concentration drops to suffi-

cell lines have been employed, including cells that ciently low levels, the cells will die. This can occur even have been genetically modified. In applications where with islets cultured and exposed to normal atmospheric tissue requirements are high, such as diabetes, the conditions, as shown in Fig. 2. The necrotic cores grow supply of tissue for immunoisolation will likely rep- in size, and the thickness of the viable rim shrinks as

Implantable biohybrid artificial organs 0 C.K. COLTON 417

Oxygen Gradient

Islet of Lange fbm;

Immui70- l~olat~on

Membrane

Blood/ Tksue

I Cel Is

\ Proteins

Components of Cellular and Humorol Immune Response

Insulin Metabolic Wastes.

??Lffcflc &la ??co2 ??Hf

Fig. 1. Transport requirements of the biohybrid artificial pancreas.

islet size increases. Conversely, as islet size decreases, the core region decreases in size, and necrotic regions are infrequent under these conditions for islet diameters less than about 150 pm.

Hypoxia can have deleterious effects on cell func-

tion. The hypoxic inhibition of second phase insulin secretion from perifused rat islets is shown in Fig. 3, where the fraction of normoxic insulin secretion rate is plotted vs. the bulk oxygen partial pressure in peri- fusion experiments. The results show that the second

Fig. 2. Rat islets cultured for 2 wk in Dulbecco’s modified Eagles medium with 10% new born calf serum (depth = 6 mm) and pOZ = 142 mm Hg. Islet diameters ranged from about 100 pm to as large as about 450 pm. Necrotic regions, visualized by staining with trypan blue, first appeared after 4-7 days, grew until 2 wk of culture, and remained the same thereafter. x25. From Dionne (20).


0 20 40 60 ” 142

BULK PERFUSATE ~02 (mm HB)

Fig. 3. Hypoxic inhibition of second phase insulin secretion from perifused rat islets following a centration from 5.5 to 16.5 mm. S14* is the second phase insulin secretion rate at bulk perfusate steady state value following a decrease of bulk pOZ to x mm Hg. From (21).

step increase in glucose con- pOZ = 142 mm Hg. S, is the

phase secretion rate begins to decrease when bulk p02 falls below about 60 mm Hg and reaches 50% of normal secretion rate at a pOZ of 27 mm Hg. When islet cell aggregates having a mean diameter of 35 pm are similarly perifused, the effect of lowered pOZ is greatly reduced because of their smaller size (20). The effect of reduced pOZ on energy-dependent processes is not limited to islet cells. For example, reduced pOZ lowers basal secretion rate of adrenaline and noradrenaline by encapsulated chromaffin cells cultured for 2 wk (27). Furthermore, reduced pOZ can also influence non- secretory cell functions. For example, the initial stages of attachment and spreading of hepatocytes on extracellular matrix are compromised at low pOZ (54). Thus, the hypoxic pOZ that undoubtedly can occur in biohybrid devices may substantially compromise cell function, even under conditions where the cells remain viable.

DEVICE DESIGN AND PERFORMANCE

In this section we consider the various bioartificial device approaches that have been investigated recently, and we examine the problem of oxygen diffusion limitations and how various solutions to this problem have evolved for different designs.

Intravascular Arteriovenous Shunt The current intravascular approach is the direct

descendent of the original concept of Chick and col-

leagues (13). It has received the longest period of study, is the most technically advanced, and has been tested extensively in large animals (52,65).

The device uses a single, coiled, tubular membrane with an internal diameter of 5-6 mm and a wall thickness of 120-140 pm. The membrane is similar to the XM50 (nominal 50,000 molecular weight cutoff) hollow fiber ultrafiltration membrane originally marketed by Amicon Corp. in the 1970s. It is fabricated from a copolymer of polyacrylonitrile and polyvinyl chloride (PAN-PVC) by a phase inversion process involving precipitation into aqueous solution of a solution of polymer in a water-miscible solvent (50). As used in the intravascular device, the membrane is asymmetric having a thin, dense retentive skin a small fraction of a micrometer thick supported on an open spongy matrix. The skin determines solute rejection and hydraulic permeability properties. The supporting matrix provides mechanical strength and determines the diffusive permeability for low molecular weight solutes. The membrane is incorporated within an acrylic housing. The islet chamber, which has a thickness of 500-750 pm, is created by the space between the membrane and the housing. The membrane cannot be sutured, so it is connected to standard PTFE graft material of the same diameter, which extends beyond the housing, and is used for anastomosis to the vascular system (Fig. 4). The islets are suspended in an extracellular gel matrix. Although the lumen is exposed to arterial blood with a pOZ of about 100 mm Hg, it has not been possible


Fig. 4. Intravascular biohybrid artificial pancreas for implantation as arteriovenous shunt. Courtesy of Claude Mullin.

to support viable islets with a tissue density greater than about 5-10% by volume.

Recent results in dogs are very promising. As of Feb- ruary, 1995, one dog has been maintained on a single patent, implanted device containing 300,000 adult porcine islets (the yield from one or two pigs) for a period of 9 mo with a 70% reduction in insulin requirements to maintain normoglycemia (48). The apparent success with immunoisolated xenotransplantation may result in part from the high rate of blood flow past the membrane which prevents immune cells from adhering to the membrane or collecting in its immediate vicinity. Phase I trials in humans are being planned. Despite the technical achievements and promise of this approach, it is possible that it may only be applied to the most un- controllable patients for two reasons. First, implantation requires major surgery that produces a permanent, irreversible break in an otherwise intact cardiovascular system. Second, it poses a possible threat of thrombo- embolism and may require some degree of permanent anticoagulation. Thus, even if clinical success is achieved, there is motivation to develop extravascular approaches which do not suffer from these problems.

Microcapsules Spherical microcapsules containing encapsulated tis-

sue have been heavily investigated. Most popular is the alginate-poly L-lysine membrane in which droplets containing cells in a solution of one weak polyelectrolyte (e.g., alginate, l-3% w/v) are precipitated in a bath of the oppositely charged polymer (e.g., poly r_-lysine) by interfacial coacervation (24,42). The resulting microcapsules have diameters ranging from 500 to 800 pm (17). A consequence of this large size, for example in the case of islets, is that the volume of the implanted preparation may be as much as two orders of magnitude greater than that of the implanted tissue.

Methods for making much smaller microcapsules have been described (25), and new techniques for prepar- ing conformal gel coatings to islets are being devel- oped (35).

Formation of microcapsules high in glucuronic acid content and low in mannuronic acid content is reported to result in improved biocompatibility and reduced fi- brosis on the external surface (60). Capsules of this for- mulation have been studied with allogeneic transplants in immunosuppressed dogs (61-63) and most recently in an immunosuppressed human (64). Encapsulated human islets (10,000 islets/kg initially, additional 5,000 islets/kg after 6 mo) were injected intraperitoneally in a diabetic patient with a functioning kidney graft who was on low-dose maintenance immunosuppression. In- sulin independence with tight glycemic control was demonstrated 9 mo after the procedure. These results demonstrate that a sufficient number of islets remained viable and functional to maintain normoglycemia. This may reflect the advantageous mass transfer situation of a very low volume fraction of islets placed within spherical domains. The disadvantages of microcapsules include their limited retrievability and possible mechanical failure of the spherical membrane. These problems may limit the utility of microcapsules in nonimmunosuppressed xenotransplantation, which has yet to be examined in depth.

Tubular Diffusion Chambers Hollow fibers or tubular membranes have the ad-

vantage that they can be retrieved, and they have been examined as an alternative to microcapsules. As compared to planar membranes, hollow fibers elicit a smaller foreign body response (71). Primary attention has focussed on the PAN-PVC ultrafiltration-type membranes similar to those used in intravascular devices. Some examples of membrane structures investigated by Cytotherapeutics Corp. are illustrated in Fig. 5. A restrictive skin may or may not be present on the lumen side, and there is an outer skin which can be smooth or fenestrated. An internal barrier can also be formed for further modification of transport properties. In general, a smooth outer skin (the surface that interfaces with host tissue) provokes little or no fibro- sis, whereas the rough fenestrated surface, which al- lows host tissue to grow into the spongy matrix, causes a substantial foreign body response (36,37,47). Further improvements in biocompatibility by reducing surface adsorption of proteins has also been investigated (59).

Initial studies with these membranes, carried out by filling the lumen with dilute suspensions of cells or islets, were unsatisfactory. Figure 6 shows an example of a PC12 cell suspension in a PAN-PVC tubular membrane. The cells aggregate into a large spheroidal mass. Extensive necrosis occurs in the center of the


Fig. 5. Structures of asymmetric ultrafiltration-type membranes prepared from copolymers of polyacrylonitrile and polyvinyl chloride (PAN-PVC) investigated for use as tubular diffusion chambers. Courtesy of Michael Lysaght.

spheroid as a result of oxygen diffusion limitations. When these anchorage-dependent cells are suspended in a gel matrix of chitosan prior to injection, they do not aggregate. Instead, they divide until they fill 20- 50% of the membrane volume in a relatively homo- geneous suspension. Nonproliferating cells also benefit from the presence of a gel matrix because they cannot

settle under gravity or aggregate. Selection of an op- timum matrix varies from one cell type to another and is likely influenced by the nature of the charge on the hydrophilic gel (e.g., alginate carries negative charge, chitosan positive charge).

A similar phenomenon occurs with islets (Fig. 7). Without extracellular matrix, islets injected at rela-

Fig. 6. Dopamine-secreting PC12 cells implanted in a rat brain in a PAN-PVC capsule without any extracellular matrix, retrieved 24 wk after implantation, and stained with tyrosine hydroxylase stain. Cells aggregate into spheroid, leading to central necrosis. Original membrane diameter about 600 pm. Courtesy of Patrick Aebischer.


tively high density aggregate into a very large mass, and most of the tissue ultimately becomes necrotic, ex- cept for a thin rim of viable tissue around the exterior of the islet (Fig. 7a). However, when the islets are prevented from contact by 1% alginate gel and limited to about 5-10% volume fraction, most of the tissue re- mains viable (Fig. 7b). At higher tissue density, viability is reduced. Although the use of narrow-bore tubular membranes with a low density of tissue suspended in alginate matrix has demonstrated maintenance of islet viability, enormous lengths of such membranes would be required for a human implant, thereby rendering the approach impractical for clinical application. Con- versely, the low tissue volume required for central nervous system applications makes this approach attractive.

An alternative for islet transplantation is to use wider bore tubes. However, the problem of cell death as a result of oxygen supply limitations, or accumula- tion of wastes or other agents, is exacerbated by substantially increased diffusion distances. Figure Sa shows viable islet tissue located near the inner membrane surface of a 2.2 mm internal diameter tubular membrane diffusion chamber retrieved from a rat peritoneal cavity after 10 mo implantation (38). Islets in the center of the chamber were histologically intact but some cell death may have occurred. In contrast, tubular membranes with a 4.8 mm internal diameter contained a large central necrotic core, as shown by cross-sectional views in Fig. 8b. Only a rim of islets remained intact within roughly 0.5-l mm of the inner surface. The white core (composition unknown) contained no viable islets. Although as much as two-thirds or more of the original implanted islet mass was dead or nonfunctional, these devices nonetheless maintained normoglycemia over many months because very large numbers of islet equivalents (about 30,000 or 60,000 islets/kg based upon 150 pm mean diameter) were implanted.

Some recent results (Fig. 9) indicating the loss of significant islet mass as a result of oxygen supply limitations in naked transplantation (7) are noteworthy, lest the reader be misled that this problem is confined to immunoisolation devices. Mouse islets were transplanted under the kidney capsule of nondiabetic, normal syngeneic mice. After 1 day (Fig. 9a), islets were surrounded with interstitial fluid and free red cells, and some islet tissue had died as evidenced by erosion of the normal islet spheroidal shape and the presence of densely heterochromatic nuclei in pale-staining cyto- plasm. After 3 days (Fig. 9b), the islets have fused into larger masses with evidence of central necrosis. After 7 days, there is no obvious cellular debris or vacant spaces, the tissue has remodelled, and small blood vessels have invaded the islet mass.

Planar Diffusion Chambers Planar diffusion chambers, preferably with high tis-

sue density, represent an attractive configuration for immunoisolation devices. Diffusion chambers prepared with parallel, flat sheet microporous membranes were employed extensively several decades ago in early studies of immune rejection during transplantation. Invari- ably, the membranes employed produced an extensive fibrotic foreign body response composed of an avascular layer of fibroblasts in a collagen matrix which re- stricted the supply of oxygen and nutrients, resulting in minimal survival of the encapsulated tissue (58).

At this point it is instructive to examine the supply of oxygen to encapsulated tissue in the idealized geom- etries of a spheroid and a planar slab to provide per- spective on the aforementioned studies with low tissue density and to gain insight on the requirements for a useful planar device. This is accomplished by use of theoretical models of oxygen reaction and diffusion in tissue for the case of oxygen consumption described by a single Michaelis Menten expression. We used the following value of reaction and transport parameters which are preliminary estimates for islet tissue: maximum reaction velocity V,,, = 3 x lo-’ mol/cm3 s, Michaelis constant, K,,, = 0.44 mm Hg, effective oxygen diffusion coefficient D = 1.6 x lop5 cm*& and Bunsen solubility coefficient CY = 1.02 PM/mm Hg (5).

Figures 10 and 11 illustrate oxygen partial pressure (~0~) profiles in tissue with p02 = 40 mm Hg at the surface, which corresponds to a typical value for the microvasculature. Figure 10 applies to spheroids of different diameter and Fig. 11 to slabs of different thickness. In both cases, p02 decreases as distance away from the surface increases. However, the rate of decrease of p02 with distance is much greater for the slab as compared to the sphere, all other parameters equal. For example, the p02 in a slab becomes essen- tially zero at the center for a thickness of 150 pm, whereas anoxia does not occur in a sphere until the diameter is 250 pm. This difference in behavior is a consequence of the different ways in which area and volume change with size in a sphere and in a slab.

This distinction becomes even more dramatic when we add an external layer between the implanted tissue and the oxygen source (blood supply). This layer would correspond to a semipermeable membrane and/or the host tissue. In this initial approximate analysis, we have assumed that the oxygen diffusivity and solubility in the external layer are the same as in the implanted tissue and that no oxygen consumption occurs in the external layer. The single implanted spheroid surrounded by a spherical external layer corresponds to the case of very low tissue density in which other spheroids are far enough away so as not to perturb the p02

Figure 7

Figure 8

(b)

(b)

(b)

Figure 9


0 50 100 150 Distance from the Surface of the Spheroid (pm)

Fig. 10. Oxygen partial pressure (PO,) profiles in tissue spheroids of different diameter (d) for the case of a 40 mm Hg surface pOZ.

profiles around the spheroid of interest. The implanted slab surrounded by a planar external layer corresponds to the opposite situation of very high tissue density in a planar system. Intermediate tissue densities would give behavior falling between these extremes.

Figures 12 and 13 show the effect of different distances from the surface of the implanted tissue to a 40 mm Hg source for a spheroid and slab, respectively. Here again, the spherical geometry is much less sensi- tive to the diffusion resistance of the external layer than is the slab. For example, for a 150 pm sphere, the p02 at the center of sphere is not anoxic, even for an external distance of 150 pm to the oxygen source. The worst case, where the oxygen source is moved to an in-

finite distance away, still retains a non zero pOZ for a distance of about 50 pm from the sphere surface. In contrast, a 150 pm layer external to a slab of only 100 pm thickness causes a very large drop in pOZ external to the surface of the tissue, and only about 10 pm from the surface is not anoxic. Only when the oxygen source is brought to within about 25 pm from the surface of the implanted tissue does the pOZ at the center of a 100 pm-thick slab stay above zero.

In order for an individual islet to benefit from mass transfer in a spherical geometry, it must feel minimal presence of the other islets, which means a very low tissue density. For cell viability to be maintained in the high tissue density slab geometry, it is essential to bring

Fig. 7. Rat islets in PAN-PVC hollow fibers with 600-m internal diameter and 80 pm wall thickness. H and E. (a) Without extracellular matrix, islets implanted in rat peritoneum for 3 wk aggregate into one large mass, leading to necrosis of most of the tissue. (b) Islets remain viable in 1% alginate matrix at low tissue density (5-10% by volume) implanted in the mouse peritoneum for 3 wk following xenotransplantation (33). The alginate gel shrinks during fixation. Courtesy of Keith Dionne.

Fig. 8. Canine islets (tissue density about 1% volume fraction in 1.2% alginate) in PAN-PVC tubular capsule implanted in peritoneum of diabetic rat. (a) Viable islets near membrane inner wall with 2.2 mm i.d. retrieved from peritoneal cavity after 307 days xenotransplantation. H and E. x220. From (38). (b) Islet-containing alginate matrix removed from 4.8 mm i.d. membrane retrieved 2 mo after xenotransplantation and sliced open. Intact islets are limited to the outer rim. Courtesy of Robert Lanza.

Fig. 9. The effect of oxygen supply limitations on naked islet syngeneic transplantation under the kidney capsule. Grafts were excised (a) 1, (b) 3, and (c) 7 days after transplantation. x 175. Sections were stained with (a) toluidine blue and eosin or (b and c) toluidine blue alone. Red cells are (a) red or (b and c) blue. Courtesy of Susan Bonner-Weir.

424 Cell Transplantation ??Volume 4, Number 4, 1995

0 20 40 60 80 100

Distance from the Surface of the Slab (pm)

Fig. 11. Oxygen partial pressure profiles in tissue slabs of different thickness (L) for the case of a 40 mm Hg surface ~0,.

the source of oxygen very close to the surface of the implanted tissue. Because oxygen supply is the limit- ing factor in maintaining viability and function of implanted tissue, maximum vascularization of adjacent

host tissue is more critical for the slab geometry than the spherical geometry.

This requirement poses great demands on the materials used in an implanted biohybrid device. The re-

40

35

30

25

20

15

10

5

0 150 100 50 0 50

Distance from the Surface of the Spheroid (pm)

Fig. 12. Oxygen partial pressure profiles inside and outside of a spheroid (d = 150 pm) for different distances (h) between the spheroid surface and an oxygen source at p0, = 40 mm Hg.


150 100 50 0 50

Distance from the Surface of the Slab (pm)

Fig. 13. Oxygen partial pressure profiles inside and outside of a slab (L = 100 pm) for different distances (h) between the slab surface and an oxygen source at ~0, = 40 mm Hg.

sponse to implanted materials can be divided into three idealized categories. The foreign body response produces an avascular layer adjacent to the membrane, within thickness typically on the order of 100 pm, that moves the oxygen source away from the surface. This response would render impossible the attainment of high tissue density in a diffusion chamber. A neutral response is one in which there is little or no adjacent fibrotic tissue but also no blood vessel growth. This kind of response may represent what has been observed, for example, in the implantation of biocom- patible microcapsules and hollow fibers or tubular membranes of PAN-PVC with a smooth outer skin, and it explains why a low tissue density has been required for cell viability with these materials. The third category is induced neovascularization, where growth and proliferation of new blood vessels near the interface minimizes diffusion distances and maximizes blood flow rate and pOZ near the interface. Under these conditions, attainment of high tissue density is, in principle, possible.

Recently, there has been reported (8) discovery of a class of microporous membranes that induce neovascularization at the material-tissue interface, referred to as close vascular structures, after 3-wk SC implantation in rats. This phenomenon could improve mass transport by bringing the microvasculature in close proximity to implanted cells. All membranes with a

nominal pore size less than 0.8 pm produced a foreign body capsule. Angiogenesis occurred in the adjacent tissue, sometimes embedded beneath a fibrotic response, when the pores were large enough for host cells to enter the interstices of the matrix. However, cell penetration alone was not sufficient for the effect because some membranes that were penetrated by cells did not cause neovascularization. Membranes that induced neovascularization were made of strands or fibers having a diameter of less than 5 pm. In contrast, non- vascularizing membranes had internal solid structures with plate-like qualities having dimensions greater than 5 pm. The invading cells retained a rounded morphol- ogy in vascularizing membranes. If cells did not enter the membrane, or if the cells that entered were able to flatten on internal structures, the material induced a foreign body response with no neovascularization. Similar results were found with microporous membranes made of cellulose acetate, mixed esters of cellulose, acrylic copolymer, and polytetrafluoroethylene (PTFE, Gore Teflon) membranes. Similar results were also reported (9) with a composite structure made by laminating a 5 pm expanded PTFE (Gore) vascularizing membrane (about 15 pm thick), which interfaces with host tissue, to a cell-impermeable 0.45 pm hydrophilic PTFE Biopore (Millipore) membrane (about 25- 30 pm thick), henceforth referred to as a microporous laminate. Vascular structures induced by the micro-


porous laminate were still present after 1.5 yr in rats. Because the Biopore membrane component of the laminate prevents passage of host cells, it could be used as an immunoisolation membrane with respect to the cellular immune response.

Figure 14 illustrates neovascularization at the host tissue-membrane interface with two examples. A cellulose acetate microporous membrane having a nominal pore size of 3 pm after 3 wk SC implantation in the rat is shown in Fig. 14a. Figure 14b is an example of the microporous laminate with the 5 pm expanded Gore PTFE membrane closest to the tissue. Both examples are from cross sections through the membrane. In each case, newly formed blood vessels were present in the vicinity of the membrane surface.

A variety of cultured cells encapsulated in planar diffusion chambers fabricated from the vascularizing microporous laminate membranes were implanted into athymic rats (10). Some encapsulated cell types, as well as rat pancreatic islets, stimulated vascularization beyond that observed in empty devices. This tissue-driven vascularization occurred only when membranes had high permeability. In membranes with low permeability, poor tissue survival was coupled with a foreign body response. It was concluded that neovascularization is induced by membrane architecture, and it is further enhanced by some implanted tissues when the membrane is sufficiently permeable to allow tissue survival during the ischemic phase of implantation. The interplay between membrane-driven and tissue-driven vascularization is poorly understood.

The laminated vascularizing membrane structure described above was fabricated into a sandwich-like device, the interior of which could be accessed through a port to inject cells (30). This ported device design also provided improved maintenance of membrane paral- lelism that helped to control the thickness of encapsulated tissue. If implanted without cells, the device integrated into the soft tissues of the host and became vascularized, forming a prevascularized organoid structure which could be separated from the adjacent tissue and was easily exteriorized for injection of tissue.

According to Fig. 13, planar diffusion chambers bearing an outer layer that induces neovascularization should be capable of immunoisolation with moderate to high tissue densities while maintaining cells in a viable state. Some examples using the microporous laminate membranes which show that this is indeed the case are depicted in Figs. 15 and 16. In this and sub- sequent examples, the 5 pm expanded PTFE membrane has bound to it tri-lobular polyester fibers (diameter about 30 ,um) for mechanical strength which extend about 125 pm into the host tissue. In Fig. 15, human fibroblasts, which may be useful for gene ther-

apy applications, were implanted in athymic rats so that immune rejection was not a problem. Viable cells filled the entire tissue compartment when its thickness was constrained to be no greater than about 250 pm (Fig. 15a). When the thickness was not constrained to this value, and the cells were allowed to continue to divide, they outgrew their oxygen supply and a necrotic core eventually formed. The maximum thickness of the tissue compartment in Fig. 15b was about 620 pm. The thickness of viable tissue, measured inward from the inner surface of the Biopore membrane, ranged from 210 to 240 ,um. These values are substantially larger than the estimate of the thickness of viable islet tissue attainable in Fig. 13, which is consistent with these fibroblasts having a rate of oxygen consumption substantially smaller than that of islets.

Figure 16 shows two examples with islets in which host animals were immunosuppressed to prevent immune rejection. About 2,000 rat islet equivalents were loaded in ported devices without any extracellular matrix and implanted in NOD mice immunosuppressed with Cyclosporine A. The mice were normoglycemic for several months prior to sacrifice. The same results were achieved using anti-CD4 antibodies (44). Fig- ure 16A shows a cross section of a device explanted after 77 days. The device was constrained to have a maximum thickness of 1 lo-120 pm for the tissue compartment. A high density of viable islet tissue was achieved. Islets were flattened and arranged themselves into two layers. Some islets aggregated into larger, flattened masses, while others did not aggregate and remained separated by connective tissue that may have been secreted by fibroblasts present in the original islet preparation. Another approach is illustrated by some very recent preliminary results in Figs. 16b and 16c in which porcine islets were first encapsulated in a conformal coating of alginate before loading into a planar diffusion chamber and implanted into triply immunosuppressed baboons. After 7 wk, a substantial fraction of the islets were viable, as illustrated in Fig. 16b by a flattened islet in a region of the tissue compartment having a 90 pm thickness and in Fig. 16c by several islets that stained positively for insulin in a region with 140 pm thickness.

PROTECTION FROM IMMUNE REJECTION

Requirements for Immunoisolation The central concept of immunoisolation is place-

ment of an immunoprotective semipermeable barrier between the host and the transplanted tissue. What does this barrier have to keep away from the tissue, and is it indeed possible to prevent immune rejection with a simple passive barrier? To answer this question,

Fig. 14. Neovascularization at the host tissue-membrane interface after 3 wk implantation in rat SC tissue. H and E. A. Sartorius 3 pm cellulose acetate membrane. B. Microporous laminate (see text for description). Delamination is artifact of histological processing. Courtesy of James Brauker.

(a) (b)

Fig. 15. Human fibroblasts (MSU 1.2) in planar diffusion chamber with microporous laminate membranes retrieved from epididymal fat pad of athymic rat 3 wk after implantation. Maximum thickness of tissue compartment: (a) 250 pm, (b) 620 pm. H and E. x62.5. The bar at right is 320 Frn long. Courtesy of Laura Martinson.

(a) (b)

Fig. 16. Islets of Langerhans in planar diffusion chambers with microporous laminate membrane. A. Rat islets implanted in ovarian fat pad of NOD mouse immunosuppressed with cyclosporine and retrieved 77 days after transplantation. H and E. x 125. From (44). Courtesy of Tom Loudovaris. B. Porcine islets implanted in omentum of normal baboon immunosuppressed with Cyclosporine A, azothioprene, and prednisone. Retrieved after 4 wk. H and E. x215. (c) Same as (b) but stained with hematoxylin and anti-insulin antibody conjugated to horseradish peroxidase. Courtesy of David Scharp.


we must consider the possible rejection pathways in immunoisolation (Fig. 17). The process may begin by diffusion of immunogenic tissue antigens across the barrier. There are several possible sources for these antigens. They may be shed from the cell surface or may be protein secreted by live cells. In addition, there could be cytoplasmic protein liberated from dead cells. Recognition and display of these antigens by antigen presenting cells initiates the cellular and humoral immune response. The former leads to activation of cytotoxic cells, macrophages, and other immune cells. Clearly, these cells must be prevented from entering the tissue compartment, a requirement that is relatively easy to meet. Potentially more serious, because it is more difficult to accomplish, is keeping out components of the humoral immune response. These include cytokines and lymphokines, for example, interleukin-1 , which can have deleterious effects on P cells, as well as the newly formed antibodies to immunogenic antigens which have leaked across the barrier. In a recent investigation, antibodies against tissue immunoisolated within a tubular diffusion chamber were present in plasma 2 to 6 wk post implantation, thereby suggest- ing that islet cell antigens crossed the membrane and stimulated antibody formation in the host (39). In addition, there may be naturally occurring antibodies, most likely IgM, to cell surface antigens (e.g., major histo- compatibility complexes) on xenografts. Antibodies produced during preexisting autoimmune disease, such as Type I diabetes, might also bind to surface antigens

on xenogeneic cells. Lastly, macrophages and certain other immune cells can secrete low-molecular weight reactive metabolites of oxygen and nitrogen including free radicals, hydrogen peroxide, and nitric oxide, that are toxic to cells in a nonspecific fashion. Most free radicals are highly reactive, have a short lifetime, and act locally where they are secreted. The extent to which these agents may play a role in causing rejection of immunoisolated tissue depends upon how far they can diffuse before they disappear as a result of chemical reaction. Calculations of penetration distances (Fig. 18) suggest that these agents can diffuse large distances if their lifetime exceeds 1 s. It has been observed that islets contained within alginate microcapsules were lysed in vitro by nitric oxide secreted by activated macrophages (68), thereby demonstrating that the toxic effects mediated by nitric oxide metabolites can take place at relatively large distances from the point where nitric oxide is secreted.

Binding of an antibody to a cell surface antigen, by itself, will not cause any problem unless the epitope is on a receptor necessary for cell function. The cytotoxic events begin if complement components pass through the membrane. Binding of the first component, Clq, to an IgM or two or more IgG molecules initiates a cas- cade culminating in formation of the membrane attack complex which can lyse a single cell. Both IgM and Clq are larger than IgG. Thus, if passage of host IgM and Clq across the barrier can be prevented, then a specific, antibody-mediated attack on the islet cells should be

POSSIBLE REJECTION PATHWAYS IN IMMUNOISOLATION: A Cimnlifiarl V;au n “rr,rp####v” ,,.a,.

, , , , , , , , , , , , , , , Li; ;lzE[ , ,,~, , , , , , , , , , , , , , Semipermeable hmunoisolation Barrier

D naKals

neactive Oxygen A 0 ??-

& Nitrooen htermedlates M _.

Cvtotoxic Cell Cellular Response - I Humoral Response-

Fig. 17. Possible rejection pathways in immunoisolation.


20

/

D= 1.8X10%m2/sec

00 L = ( D t )“*

Lifetime, t (set)

Fig. 18. Dependence of penetration depth on lifetime for a low molecular weight solute.

prevented. If the alternate complement pathway is activated and not inhibited, then passage of C3 across the membrane must also be prevented. Although its molecular weight is 410,000, roughly half that of IgM, both IgM and Clq have a smallest dimension of about 30 nm. C3 has a molecular weight of about 200,000 and is smaller than Clq. Both Clq and IgM should be completely retained by a membrane with a maximum pore diameter of 30 nm. In contract with extracellular fluid, the pores of a hydrophobic membrane are likely to be coated with a monolayer of protein, say roughly 10 nm thick. Thus, pores with diameters of about 50 nm would be needed to allow Clq and IgM to pass through.

Oxygen Glucose

Free Radicals

Reactive Oxygen and Nitrogen Metabolites

The mechanism(s) that may play a role in rejection of immunoisolated tissue are, in general, unknown, and will likely depend upon the specific types of cells present, the species, and its phylogenetic distance from humans. Since the earliest work on immunoisolation, it has been tacitly assumed that a membrane with a nominal 50,000-100,000 molecular weight cutoff, which should retain IgG, would be adequate to provide protection. It is now recognized, however, that this is an oversimplification. Some of the solutes of interest in immunoisolation are shown as a function of molecular weight in Fig. 19. Oxygen, glucose, growth factors, and most likely albumin (carries free fatty acids) and transferrin (carries iron) are required for viability

I I I I I 1 10 102 103 lo4 105 106

Molecular Weight

Fig. 19. Molecular weight spectrum in immunoisolation. Molecules which should pass the immunoisolation barrier (e.g., nutrients, secreted products) are in italics; all other molecules may be deleterious to implanted tissue.


and function of implanted tissue, although the quan- titative requirements of the protein molecules for specific tissues are largely unknown. Permeability to secreted products, such as insulin, must be high enough for therapeutic efficacy. All of the other molecules shown in Fig. 19 are potentially deleterious and are associated with damage mechanisms. If free radicals and/or reactive oxygen and nitrogen intermediates pose a significant problem, then no passive membrane barrier will be able to provide immunoisolation, and some other approach (e.g., scavenging of free radicals, local immunosuppression) will be necessary. If complete retention of IgG, or even Clq and IgM, coupled with passage of albumin and transferrin is required, then the problem is still difficult because of the size discrim- ination of real membranes.

For purposes of illustration, we consider the asymmetric ultrafiltration-type membranes. Figure 20 is an en face photomicrograph of the retentive skin of a 50,000 nominal molecular weight cutoff membrane similar to the PAN-PVC membranes used extensively in immunoisolation and Fig. 21 its pore size distribution plotted in terms of both relative pore number and relative pore area. The pore size distribution is broad.

Fig. 20. Field emission scanning electron micrograph show- ing en face view of retentive skin on lumen surface of asymmetric ultrafiltration membrane having nominal 50,000 molecular weight cutoff (Romicon PM50, polysulfone; x 100).

r 1.0

0.8

0.8

0.4

0.2

0 20 40 60 80 PORE DIAMETER (nm)

Fig. 21. Pore size distribution in skin of nominal 50,000 molecular weight cutoff ultrafiltration membrane shown in Fig. 20.

Despite its nominal cutoff properties, there is a long tail representing large pores that may be viewed as de- fects. For example, about 1% of the pores, representing about 5% of the total pore area, are larger than 50 nm. These pores would permit passage of antibodies and complement at sufficient rates to present problems (17) if antibody-mediated mechanisms are important.

Experimental Studies of Immunoisolation: Allografts

Immunosuppression regimes that act largely or en- tirely on the cellular immune response have been successful in ameliorating immune rejection of allogeneic tissue in humans. It is therefore reasonable to hypoth- esize that prevention of contact with the cellular immune system of the host would allow immunoisolation of allografts. This issue has received much less attention with nonimmunosuppressed hosts than xenografts because of the limited supply of primary human tissue. However, allogeneic cells are of interest in applications, such as gene therapy, that involve genetic manipulation. An example directed towards that application is illustrated in Fig. 22 which shows baboon fibroblast-like cells implanted subcutaneously in a ba-


boon within a planar diffusion chamber with micro- ties. For example, the presence of a charge on a hydro- porous laminate membranes and retrieved after 3 wk. gel leads to a substantial reduction in the effective When the membrane was punctured (Fig. 22a), thereby diffusion coefficient and equilibrium partition coef- allowing access to host cells, the tissue was destroyed. ficient of like-charged proteins (29). Conversely, the However, with the intact membrane (Fig. 22b), the tis- presence of opposite charges on the gel and diffusing sue remained viable with no signs of immune rejection. protein could cause the gel to act as a sink by adsorb- The same result was observed with tissue retrieved af- ing the protein. Indeed, in at least one case, that of rat ter 10 wk. Although it is not known what role the host islets implanted in C57 mice, the same immunoisola- humoral immune response might play over much lon- tion result was obtained when the islets were encap- ger time scales, these results clearly demonstrate suc- sulated in the same alginate matrix but without the cessful allogeneic immunoisolation for many months. PAN-PVC membrane (34).

Experimental Studies of Immunoisolation: Xenografts

Immunoisolation of xenogeneic tissue is more difficult than with allogeneic tissue because of the complex role played by the humoral immune response in the former case. Prevention of immune rejection of xenogeneic islets in small animals has been achieved with microcapsules (67) and planar diffusion chambers with microporous laminate membranes (44) together with immunosuppression or immunomodulation. In both of these studies, inhibition of CD4+ helper T cells was one of several successful approaches (45). It has been suggested that, in the case of islet xenotransplantation (57), CD4+ macrophages actually play the critical role, rather than T cells.

Some success has been achieved in nonimmunosuppressed host animals with devices using PAN-PVC membranes. Islet xenograft immunoisolation has been reported in the aforementioned studies with intravascular devices and small diameter tubular diffusion chambers in C57 mice. The small tubular devices have also been successful with various tissues in a variety of animals when implanted in the central nervous system. It is possible that the PAN-PVC membrane has permeability properties which alone account for prevention of rejection. However, a number of other factors likely play a critical role, thereby confounding this conclu- sion. Because the membrane is in contact with flow- ing blood in the intravascular device, the implanted tissue is not subject to the inflammatory response which can surround a device placed in soft tissue. The central nervous system is thought to be an immunolog- ically privileged site, leading to a much weaker immune response. Two common characteristics of all of the studies carried out with PAN-PVC membranes in ei- ther the intravascular or extravascular tubular diffusion chamber configuration are that the tissue density is very low and the tissue is suspended in a gel matrix. The low tissue density leads to a low flux of shed antigens. In addition, the large diffusion distances leads to a lower local concentration of humoral agents. The gel matrix itself may have immunoprotective proper-

Immunoisolation of tissue at high density poses a more difficult problem than low density. A recent study has examined the role that membrane properties might play in achieving xenograft immunoisolation with high density tissue in planar diffusion chambers (49). Xenotransplantation experiments were carried out with cellulose acetate membranes that had substantially reduced permeabilities to high molecular weight solutes as compared to the microporous laminate membranes. The cellulose acetate membranes were cast from 15% and 20% solids concentration and were bonded to the vascularizing 5 pm expanded PTFE membranes which formed the interface with tissue. Figure 24 is a plot of the ratio of the effective diffusivity in each membrane to that in water as a function of solute molecular weight for the two cellulose acetate membranes and for a typical PAN-PVC membrane (22) for com- parison. The effective diffusivity D, was calculated from the measured diffusive permeability and an estimate of the total thickness L according to D, = P, L. The relative diffusivity ratio decreased with increasing molecular weight and dropped sharply for molecular weights above about 20,000. In this range, the 20% cellulose acetate membrane was roughly an order of magnitude lower in effective diffusivity.

Figure 23 shows the results of experiments with implanted rat embryonic lung tissue. Isografts (Figs. 23a-c) and xenografts (Figs. 23d-f) were implanted in planar diffusion chambers with microporous laminate membranes which do not protect from the humoral immune response (Figs. 23a and d), 15% cellulose acetate membranes (Figs. 23b and e), and 20% cellulose acetate (Figs. 23c and f) membranes. The nature of both the graft and the membrane influenced the extent of vascularization as assessed by close vascular structures, the host immune response, and the graft tissue survival. Vascularization of the isograft was maximal in the unprotected (microporous laminate) state and decreased with decreasing membrane permeability. With xenografts, vascularization was negligible when unprotected and increased slightly with both cellulose acetate membranes. The host cellular immune response to the xenograft which surrounded the device was strong in

432

(a)

Cell Transplantation 0 Volume 4, Number 4, 1995

(b)

Fig. 22. Baboon fibroblast-like cells from skin biopsies were grown in culture, encapsulated in planar diffusion chambers with microporous laminate membranes, implanted subcutaneously, and retrieved after 3 wk. H and E. x62.5. (a) Device with per- forated membrane. (b) Intact device. Courtesy of Tom Loudovaris.

(a)

Fig. 23. Isografts (Lewis rat fetal lung tissue) and xenografts (ICR mouse fetal lung tissue) in planar diffusion chambers were implanted in epididymal fat pads of Lewis rats and retrieved after 3 wk. (a-c) isografts; (d-f) xenografts. (a and d) microporous laminate membranes. (b and e) 15% cellulose acetate hydrogel membrane. (c and f) 20% cellulose acetate hydrogel membrane. H and E. ~62.5. From (49). Courtesy of Laura Martinson.


3 0 lo-* 1 s t .E > .-

2 E

10” Y-O- Asymmetric UF

n Lz8Opm

.$ 10-4r (Dionne et al.)

3 :- 15% CA Hydrogel

Molecular Weight

Fig. 24. Ratio of effective diffusivity in membrane to that in water as a function of solute molecular weight for three immunoisolation membranes: PAN-PVC asymmetric ultrafiltration-type membrane; 15% and 20% cellulose acetate hydrogel membranes laminated to 5 pm expanded PTFE (Gore) membrane.

the unprotected state, somewhat diminished with 15% cellulose acetate, and substantially weakened to be the same as the isograft with 20% cellulose acetate, pre- sumably because the least permeable membrane reduced the amount and/or size of xeno-antigens released

from the implant. Graft tissue survival was good for all isografts, which showed maintenance of differen- tial phenotype (ciliated epithelia). The xenograft was destroyed in the unprotected state. Survival was good with the 15% cellulose acetate but was compromised somewhat with the least permeable 20% cellulose acetate membrane.

These results demonstrate a complex interplay between nutrient and oxygen supply to implanted tissue, efflux of antigen from the implanted tissue to the host, and the consequent stimulation of an immune response against the implanted tissue. The higher permeability 15% cellulose acetate membranes did allow antigens to escape and stimulate a local immune response. None- theless, implanted tissue survival was good, and the tissue was protected from the host immune response (or that response was too weak to damage the tissue). The lower permeability 20% cellulose acetate membranes prevented the local host immune response from occurring, but the survival of the implanted tissue was compromised, possibly by limitations of nutrient supply.

One last example of some recent preliminary results is shown in Fig. 25 in which porcine islets encapsulated in a conformal alginate coating were implanted in a nonimmunosuppressed baboon in a planar diffusion chamber with the same 15% cellulose acetate membrane as employed in Fig. 23. The survival of these islets retrieved after 4 wk implantation, while anec- dotal, suggests promise for successful immunoisolation with planar diffusion chambers with appropriate choice of immunoisolating membrane.

Acknowledgments-This work was supported in part by NIH Grant HD3 1443-02. The author is indebted to the following people who provided unpublished photographs for use in this manuscript:

(4 (b)

Fig. 25. Porcine islets implanted in planar diffusion chambers with 15% cellulose acetate membranes, in omentum of normal, nonimmunosuppressed baboon and retrieved after 4 wk. (a) Intact islet. (b) Islet with small necrotic core. H and E. x300. Courtesy of David &harp.


Keith Dionne (Alza), James Brauker, Robert Johnson, Tom Loudovaris, and Laura Martinson (Baxter Healthcare), Michael Lysaght (Brown Univ.), Claude Mullin and Barry Solomon (W.R. Grace), Gordon Weir and Susan Bonner-Weir (Joslin Diabetes Cen- ter), Ore Hegre and David Scharp (Neocrin), and Patrick Aebischer (Univ. of Lausanne).

1.

2.

3.

4.

5.

6.

7. 8.

9.

10.

11.

12.

REFERENCES

Aebischer, P.; Ip, T.K.; Miracoli, L.; Galletti, P.M. Re- nal epithelial cells grown on semipermeable hollow fibers as a potential ultrafiltrate processor. Trans. Am. Sot. Artif. Intern. Organs. 35:96-102; 1987. Aebischer, P.; Tresco, P.A.; Winn, S.R.; Greene, L.A.; Jaeger, C.B. Long-Term cross-species brain transplantation of a polymer-encapsulated dopamine-secreting cell line. Exp. Neural. 111:269-275; 1991. Aebischer, P.; Wahlberg, L.; Tresco P.A.; Winn, S.R. Macroencapsulation of dopamine secreting cells by co- extrusion with an organic polymer solution. Biomateri- als 12:50-60; 1991. Aebischer, P.; Goddard, M.; Tresco, P.A. Cell encapsulation for the nervous system. In: Goosen, M.F.A., ed. Fundamentals of animal cell encapsulation and immobilization. Boca Raton: CRC Press; 1992:197-224. Avgoustiniatos, S. Oxygen diffusion and consumption in islets of Langerhans. Ph.D. Thesis. Massachusetts In- stitute of Technology, Cambridge, MA (in preparation). Bellamkonda, R.; Aebischer, P. Review: Tissue engineering in the nervous system. Biotech. Bioeng. 43:543-554; 1994. Bonner-Weir, S. Personal communication. Brauker, J.H.; Martinson, L.A.; Young, S.; Johnson, R.C. Neovascularization at a membrane-tissue interface is dependent on microarchitecture. Abstracts, Fourth World Biomaterials Congress. Berlin, FRG, April 24- 28, p. 685; 1992a. Brauker, J.H.; Martinson, L.A,; Carr-Brendel, V.E.; Johnson, R.C. Neovascularization of a PTFE membrane for use as the outer layer of an immunoisolation device. Abstracts, Fourth World Biomaterials Congress. Berlin, FRG, April 24-28, p. 676; 1992b. Brauker, J.H.; Martinson, L.A.; Hill, R.S.; Young, S.K.; Carr-Brendel, V.E.; Johnson, R.C. Neovascular- ization of immunoisolation membranes: The effect of membrane architecture and encapsulated tissue. Ab- stracts, First International Congress of Cell Transplan- tation Society. Pittsburgh, PA, June 13-15, p. 163; 1992~. Brauker, J.; Dwarki, V.; Carr-Brendel, V.; Nijjar, T.; Vergoth, C.; Chen, R.; Cohen, L.; Danos, 0.; Mulligan, R.; Johnson, R.C. Expression of human factor IX in rats and mice from cells implanted within an immunoisolation device. Abstracts, Keystone Symposium of Gene Therapy and Molecular Medicine. Mar. 27-31, 1995. Chang, P.L.; Shen, N.; Westcott, A.J. Delivery of recombinant gene products with microencapsulated cells in vivo. Hum. Gene Ther. 4:433-440; 1993.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22. 23.

24

25.

26.

27.

28.

29.

Chick, W.L.; Like, A.A.; Lauris, V.; Galletti, P.M.; Richardson, P.D.; Panol, G.; Mix, T. W.; Colton, C.K. A hybrid artificial pancreas. Trans. Am. Sot. Artif. In- tern. Organs. 21:8-14; 1975. Christenson, L.; Aebischer, P.; Galletti, P.M. Encapsu- lated thymic epithelial cells as a potential treatment for immunodeficiencies. Trans. Am. Sot. Artif. Intern. Or- gans. 34:681-686; 1988. Christenson, L.; Dionne, K.E.; Lysaght, M.J. Biomed- ical applications of immobilized cells. In: Gossen, M.F.A., ed. Fundamentals of animal cell encapsulation and immobilization. Boca Raton: CRC Press; 1992: 7-41. Cieslinski, D.A.; Humes, H.D. Tissue engineering of a bioartificial kidney. Biotech. Bioeng. 43:678-681; 1994. Colton, C.K.; Avgoustiniatos, E.S. Bioengineering in development of the hybrid artificial pancreas. ASME J. Biomech. Eng. 113:152-170; 1991. Colton, C.K. The engineering of xenogeneic islet transplantation by immunoisolation. Diab. Nutr. Metab. 5: 145-149; 1992. Colton, C.K. Engineering issues in islet immunoisolation. In: Lanza, R.P.; Chick, W.L., eds. Pancreatic islet transplantation: vol. III. Immunoisolation of pancreatic islets. Vol. III, New York: R.G. Landes Co.; 1994:13-19. Dionne, K.E. Effect of hypoxia on insulin secretion and viability of pancreatic tissue. Ph.D. Thesis. Massachu- setts Institute of Technology, Cambridge, MA, 1989. Dionne, K.E.; Colton, C.K.; Yarmush, M.L. Effect of hypoxia on insulin secretion by isolated rat and canine islets of Langerhans. Diabetes 42: 12-21; 1993. Dionne, K.E. Personal communication. Emerich, D.F.; Winn, S.R.; Christenson, L.; Palmatier, M.A.; Gentile, ET.; Sanberg, P.R. A novel approach to neural transplantation in Parkinson’s disease: Use of polymer encapsulated cell therapy. Neurosci. Biobehav. Rev. 16:437-474; 1992. Goosen, M.F.A.; Oshea, G.M.; Gharapetian, H.M.; Chou, S.; Sun, A.M. Optimization of microencapsulation parameters: Semipermeable microcapsules as a bioartificial pancreas. Biotech. Bioeng. 27:146-150; 1985. Halle, J.-P.; Leblond, EA.; Pariseau, J.-F.; Jutras, P.; Jutras, P.; Brabant, M.-J.; Lepage, Y. Studies on small (~300 pm) microcapsules: II-Parameters governing the production of alginate beads by high voltage electrostatic pulses. Cell Transplant. 3:365-372; 1994. Hoffman, D.; Breakfield, M.; Short, P.; Aebischer, P. Transplantation of a polymer encapsulated cell line genetically engineered to release NGF. Exp. Neurol. 121: 100-106; 1993. Holland, L.; Cain, B.M.; Dionne, K.E. Effect of reduced pOZ on viability and function of encapsulated bovine adrenal chromaffin cells. Abstracts, ASAIO 23:23; 1994. Hymer, W.C.; Wilbur, D.L.; Page R.; Hibbard, E.; Kel- sey, R.C.; Hatfield, J.M. Pituitary hollow fiber units in vivo and in vitro. Neuroendocrinology 32:339-349; 1981. Johnson, E.M.; Berk, D.A.; Jain, R.K.; Deen, W.M.


Diffusion and partitioning of proteins in charged aga- rose gels. Biophys. J. 681561-1568; 1995.

30. Johnson, R.C.; Carr-Brendel, V.; Martinson, L.; Neu- enfeldt, S.; Young, S.; Vergoth, C.; Hodgett, D.; Maryanov, D.; Jacobs, S.; Hill, R.; Thomas, T.J.; Brauker, J. An organoid structure for the implantation of genetically engineered cells and pancreatic islets. Ab- stracts, Second International Congress of Cell Trans- plant Society. Minneapolis, MN, May l-4, 1994. (Cell Transplant. 3:221; 1994).

31. Joseph, J.M.; Goddard, M.B.; Mills, J.; Padrun, V.; Zurn, A.; Zielinski, B.; Favre, J.; Gardaz, J.P.; Mosimann, F.; Sagen, J.; Christenson, L.; Aebischer, P. Transplantation of encapsulated bovine chromaffin cells in the sheep subarachnoid space: A preclinical study for the treatment of cancer pain. Cell Transplant. 3:355- 364; 1994.

32. Koo, J.; Chang, T.M.S. Secretion of erythropoietin from microencapsulated rat kidney cells. Preliminary results. Int. J. Artif. Organs 16:557-560; 1993.

33. Lacy, P.E.; Hegre, O.H.; Gerasimide-Vazeou, A.; Gen- tile, F.; Dionne, K.E. Subcutaneous xenografts of rat islets in acrylic copolymer capsules maintain normoglycemia in diabetic mice. Science 254:1782-1784; 1991.

34. Lacy, P.E. Personal communication. 35. Lamberti, F. Personal communication. 36. Lanza, R.P.; Borland, K.M.; Staruk, J.E.; Appel, M.C.;

Solomon, B.A.; Chick, W.L. Transplantation of encapsulated canine islets into spontaneously diabetic BB/Wor rats without immunosuppression. Endocrinology 13 1: 637-642; 1992.

37. Lanza, R.P.; Sullivan, S.J.; Chick, W.L. Perspectives in diabetes. Islet transplantation with immunoisolation. Di- abetes 41:1503-1510; 1992.

38. Lanza, R.P.; Beyer, A.M.; Staruk, J.E.; Chick, W.L. Biohybrid artificial pancreas. Long-term function of dis- cordant islet xenografts in streptozotocin diabetic rats. Transplantation 56:1067-1072; 1993.

39. Lanza, R.P.; Beyer, A.M.; Chick, W.L. Xenogeneic humoral responses to islets transplanted in biohybrid diffusion chambers. Transplantation 57:1371-1375; 1994.

40. Lanza, R.P.; Chick, W.L. Medical Intelligence Unit. Pancreatic islet transplantation: Volume I. Procurement of pancreatic islets, Austin, TX: R.G. Landes; 1994.

41. Lanza, R.P.; Chick, W.L. Medical Intelligence Unit. Pancreatic islet transplantation: Volume III. Immuno- isolation of pancreatic islets, Austin, TX: R.G. Landes; 1994a.

42. Lim, F.; Sun A.M. Microencapsulated islets as bioartificial endocrine pancreas. Science 210:980-982; 1980.

43. Liu, H.W.; Ofosu, EA.; Chang, P.L. Expression of human factor IX by microencapsulated recombinant fibroblasts. Hum. Gene Ther. 4:291-301; 1993.

44. Loudovaris, T.; Maryanov, D.; Young, S.; Jacbos, S.; Martinson, L.; Neuenfeldt, S.; Brauker, J.; Johnson, R. Correction of diabetic nod mice implanted with rat islets in immunoisolation devices. Abstracts, Second Inter- national Congress of Cell Transplant Society. Minneap-

olis, MN, May l-4, 1994, (Cell Transplantation 3:247; 1994a).

45. Loudovaris, T.: Charlton, B.; Mandel, T. CD4+ T cell mediated destruction of xenografts within cell-impermeable membranes in the absence of CD8+ T cells and B cells. Transplantation (in press).

46. Lysaght, M.J.; Frydel, B.; Emerich, D.; Winn, S. Re- cent progress in immunoisolated cell therapy. J. Cellu- lar Biochem. 56:196-203; 1994.

47. Lysaght, M.J. Personal communication. 48. Maki, T. Personal communication. 49. Martinson, L.; Pauley, R.; Brauker, J.; Boggs, D.;

Maryanov, D.; Sternberg, S.; Johnson, R.C. Protection of xenografts with immunoisolation membranes. Ab- stracts, Second International Congress of Cell Trans- plant Society, Minneapolis, MN, May l-4, 1994. (Cell Transplant. 3:249; 1994).

50. Michaels, A.S. High flow membranes. US patent 3, 615,024; 1971.

51. Mikos, A.G.; Papadaki, M.G.; Kouvroukoglou, S.; Ishaug, S.L.; Thomson, R.C. Mini-review: Islet transplantation to create a bioartificial pancreas. 43:673-677; 1994.

52. Monaco, A.P.; Maki, T.; Ozato, H.; Carretta, M.; Sul- livan, S.J.; Borland, K.M.; Mahoney, M.D.; Chick, W.L.; Muller, T.E.; Wolfrum, J.; Solomon, B. Trans- plantation of islet allografts and xenografts in totally pancreatectomized diabetic dogs using the hybrid artificial pancreas. Ann. Surg. 214:339-362; 1991.

53. Reach, G.; Poussier, P.; Sausse, A.; Assan, R.; Itoh, M.; Gerich, J.E. Functional evaluation of a bioartificial pancreas using isolated islets perifused with blood ultrafiltrate. Diabetes 30:296-301, 198 1.

54. Rotem, A.; Toner, M.; Bhatia, S.; Foy, B.D.; Tomp- kins, R.G.; Yarmush, M.L. Oxygen is a factor determin- ing in vitro tissue assembly: Effects on attachment and spreading of hepatocytes. Biotech. Bioeng. 43:654-660; 1994.

55. Rozga, J.; Morsiani, E.; LePage, E.; Moscioni, A.D.; Giordio, T.; Demetriou, A.A. Isolated hepatocytes in a bioartificial liver: A single group view and experience. Biotech. Bioeng. 43:645-653; 1994.

56. Sagen, J. Chromaffin cell transplants for alleviation of chronic pain. ASAIO J. 38:24-28; 1992.

57. Satake, M.; Korsoren, 0.; Ridderstad, A.; Karlsson- Parra, A.; Wallgren, A.-C.; Moller, E. Immunological characteristics of islet cell xenotransplantation in humans and rodents. Immunological Reviews (141): 191-204, 1994.

58. Scharp, D. W.; Mason, N.S.; Sparks, R.E. Islet immunoisolation: The use of hybrid artificial organs to prevent islet tissue rejection. World J. Surg. 8:221-229; 1984.

59. Shoichet, M.S.; Winn, S.R.; Athavale, S.; Harris, J.M.; Gentile, ET. Poly(ethylene oxide)-grafted thermoplas- tic membranes for use as cellular hybrid bio-artificial organs in the central nervous system. Biotech. Bioeng. 43:563-572; 1994.

60. Soon-Shiong, P.; Otterlei, M.; Skjak-Braek, G.; Smids-


rod, 0.; Heintz, R.; Lanza, R.P.; Espevik, T. An immu- nologic basis for the fibrotic reaction to implanted microcapsules. Transplant Proc. 23:758-759; 1991.

61. Soon-Shiong, P.; Feldman, E.; Nelson, R.; Komtebedde, J.; Smidsrod, 0.; Skjak-Break, G.; Espevik, T.; Heintz, R.; Lee, M. Successful reversal of spontaneous diabetes in dogs by intraperitoneal microencapsulated islets. Transplantation 54:769-774; 1992.

62. Soon-Shiong, P.; Feldman, E.; Nelson, R.; Komtebedde J.; Smidsrod, 0.; Skjak-Braek, G.; Espevik, T.; Heintz, R.; Lee, M. Successful reversal of spontaneous diabetes in dogs by intraperitoneal microencapsulated islets. Transplantation 54769-774; 1992.

63. Soon-Shiong, P.: Feldman, E.; Nelson, R.; Heintz, R.; Yao, Q.; Yao, Z.; Zheng, T.; Merideth, N.; Skajak- Braek, G.; Espevik, T.; Smidsrod, 0.; Sandford, P. Long-term reversal of diabetes by the injection of immu- noprotected islets. Proc. Natl. Acad. Sci. USA 90:5843- 5847; 1993.

64. Soon-Shiong, P.; Heintz, R.E.; Merideth, N.; Yao, Q.X.; Yao, Z.; Zheng,T.; Murphy, M.; Moloney, M.K.; Schmehl, M.; Harris, M.; Mendez, R.; Mendez, R.; San- ford, P.A. Insulin independence in a type 1 diabetic patient after encapsulated islet transplantation. Lancet 343:950-951; 1994.

65. Sullivan, S.J.; Maki, T.; Borland, K.M.; Mahoney, M.D.; Solomon, B.A.; Muller, T.E.; Monaco, A.P.; Chick, W.L. Biohybrid artificial pancreas: Long-term implantation studies in diabetic, pancreatectomized dogs. Science 252:718-721; 1991.

66. Tresco, P.A.; Winn, S.R.; Tan, S.; Jaeger, C.B.;

Greene, L.A.; Aebischer, P. Polymer encapsulated PC12 cells: Long term survival and associated reduction in le- sion induced rotational behavior. Transplantation 1:255- 264; 1992.

67. Weber, C.J.; Reemstma, K. Microencapsulation in small animals-Xenografts. In: Lanza, R.P.; Chick, W.L., eds. Pancreatic islet transplantation: vol. III. Immuno- isolation of pancreatic islets. New York: R.G. Landes co.; 1994:59-79.

68. Wiegand, F.; Kroncke, K.-D.; Kolb-Bachofen, V. Macrophage-generated nitric oxide as cytotoxic factor in destruction of alginate-encapsulated islets. Transplan- tation 56:1206-1212; 1993.

69. Winn, S.R.; Tresco, P.A.; Zielinski, B.; Greene, L.A.; Jaeger, C.B.; Aebischer, P. Behavioral recovery following intrastriatal implantation of microencapsulated PC12 cells. Exp. Neurol. 113:322-329; 1991.

70. Winn, S.R.; Hammang, J.P.; Emerich, D.F.; Lee, A.; Palmiter, R.D.; Baetge, E.E. Polymer-encapsulated cells genetically modified to secrete human nerve growth factor promote the survival of axotomized septal choliner- gic neurons. Proc. Natl. Acad. Sci. USA 91:2324-2328; 1994.

71. Woodward, SC How fibroblasts and giant cells encap- sulate implants: Considerations in design of glucose sen- sors. Diabetes Care 5:278-281; 1982.

72. Yang, M.B.; Vacanti, J.P.; Ingber, D.E. Hollow fibers for hepatocyte encapsulation and transplantation: Stud- ies of survival and function in rats. Cell Transplant. 3: 373-385; 1994.

implantable biohybrid artificial organs

Documents