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INTRODUCTION

CELL MICROENCAPSULATION in a semipermeable mem-brane has been a long-standing, but in some respects

unsuccessful, approach to enabling cell transplanta-tion without immune consequences. The capsule wall,whether it be alginate–polylysine or poly(hydroxyethylmethacrylate-co-methyl methacrylate) (HEMA–MMA),is intended to isolate the transplanted allogeneic or xeno-geneic cells from the host’s immune system and therebyenable the long-term survival of the transplanted cells despite the “hostile” immune environment. The algi-nate–polylysine system pioneered by Sun has been themost successful. Sun et al. have reported1 that encapsu-lated porcine islets restored normoglycemia in sponta-neously diabetic monkeys for as long as 2 years, and pre-

liminary clinical data have implied a reasonable degreeof safety associated with these capsules in human dia-betics.2 Unfortunately, the mechanical fragility of thesecapsules and other reproducibility issues have hamperedthe further development of this system for islet trans-plantation or other applications.

We have focused on using a thermoplastic syntheticpolyacrylate copolymer, HEMA–MMA (75 mol%HEMA) to microencapsulate cells. HEMA–MMA formsa soft, tough polymer membrane with adequate perme-ability to small molecules and nearly complete exclusionof larger proteins (.150 kDa). We have had much suc-cess in vitro3: almost any cell type can be encapsulatedand their viability maintained (in vitro) despite the ex-posure to nonaqueous solvents (polyethylene glycol 200or triethylene glycol) and the shear forces implicit to mi-

TISSUE ENGINEERINGVolume 9, Number 5, 2003© Mary Ann Liebert, Inc.

Viability of Hydroxyethyl Methacrylate–Methyl Methacrylate-Microencapsulated PC12 Cells after Omental Pouch Implantation within Agarose Gels

A.J. FLEMING and M.V. SEFTON, Ph.D.

ABSTRACT

Hydroxyethyl methacrylate–methyl methacrylate (HEMA–MMA, 75 mol% HEMA). Microcapsulescontaining viable PC12 cells (as an allogeneic transplant model) were implanted into omental pouchesin Wistar rats. Two different capsule preparations were tested, based on differences in polymer so-lutions during extrusion: 10% HEMA–MMA in TEG, and 9% HEMA–MMA in TEG with 30%poly(vinyl pyrrolidone) (PVP). The omental pouch proved to be an ideal transplant site in terms ofimplantation, recovery, and blood vessel proximity (nutrient supply). To minimize the fibrous over-growth and damaged capsules previously seen on implantation of individual capsules, agarose gelswere used to embed the capsules before implantation. Cells proliferated within the microcap-sule–agarose device during the first 7 days of implantation, but overall cell viability declined overthe 3-week period, when compared with similar capsules maintained in vitro. Nonetheless, approx-imately 50% of the initial encapsulated cells were still viable after 3 weeks in vivo. This approachto HEMA–MMA microcapsule implantation improved cell viability and capsule integrity after 3weeks in vivo, compared with capsules implanted without agarose.

Department of Chemical Engineering and Applied Chemistry, Institute of Biomaterials and Biomedical Engineering, Univer-sity of Toronto, Toronto, Ontario, Canada.

croencapsulation. After encapsulation, many cells willgrow but this is dependent on the presence of coencap-sulated matrix (e.g., Matrigel or collagen) and the par-ticular nature of the cell under study. We have had, how-ever, little success in vivo.

Microencapsulated PC12 cells were implanted into thestriata of brains of rats with 6-hydroxydopamine lesionsof the substantia nigra.4 Although the behavioral effectsof the lesions were not reversed (presumably because aninadequate number of cells was transplanted), the im-planted cells remained viable and expressed tyrosine hy-droxylase in vivo for at least 3 weeks. On the other hand,human hepatoma (HepG2) and rat hepatoma (H4IIEC3)cells were microencapsulated and implanted into rats, inorder to provide xenograft and allograft transplant mod-els, respectively.5 As a preferred site for implantation, therat omentum was folded and sutured into a pouch, pro-viding localization and retrievability for capsule im-plants. Implantation into the peritoneal cavity and sub-cutaneous sites was also investigated. Viability waslimited with both of these cell lines in vivo; the rat cellssurvived for up to 1 week, but the human cells lost via-bility within 4 days. This more rapid failure of thexenograft suggested an immune component to the failuremechanism. Both inflammatory and immune cells wereidentified at the site of both transplant models.5

Empty HEMA–MMA microcapsules or HEMA–MMA films appear to be biocompatible: there are fewcells attached and few, if any, inflammatory cells and al-most no fibrotic reaction in the surrounding tissue. Onthe other hand, there was a more extensive inflammatoryreaction to HEMA–MMA capsules that contained cells.5

Some of what did occur appears to be related to the pres-ence of adsorbed protein6 (a carryover from the incuba-tions in serum-containing medium before implantation)and some to the surgical manipulations. Sham direct im-plantation surgeries generated the same level of macro-phage activation as did implantation with capsules;omental pouch surgeries are presumably even more acti-vating. In a syngeneic model (encapsulated L929 cellsdirectly into the peritoneal cavity of C3H mice), incuba-tion with xenogeneic horse serum caused the formationof a more pronounced fibrous tissue around the micro-capsules.7 On the other hand, microcapsules that wereembedded in 4% (w/v) SeaPlaque agarose gel and im-planted intraperitoneally in C3H mice maintained a via-bility of approximately 50% of the L929-SEAP cells 21days after implantation.7 The agarose prevented the cap-sules from clumping into a large mass of deformed anddamaged capsules. The agarose provided a means for as-sessing the fate of encapsulated cells in vivo withoutmany of the factors that have confounded previous stud-ies. Here we explore the use of an agarose gel disk in theomental pouch and with an allogeneic cell (PC12) model.

A secondary purpose was to investigate two HEMA–

FLEMING AND SEFTON

MMA capsule formulations. We found previously8 thatsignificant increases in permeability to horseradish per-oxidase (HRP, 40 kDa) were achieved by both de-creasing the concentration of HEMA–MMA in solvent(triethylene glycol, TEG), and by introducing poly(vinylpyrrolidone) (PVP) to the polymer solution as a “pore-forming agent.” These findings led us to compare a“standard” formulation, 10% HEMA–MMA polymerdissolved in TEG (not polyethylene glycol 200) and 9%HEMA–MMA in TEG with 30% PVP (w/w withHEMA–MMA). On the basis of the previous work, cap-sules prepared with these two materials should differ inpermeability to HRP by approximately two orders ofmagnitude. We expected that this difference in capsulepermeability to HRP should enhance cell viability.

MATERIALS AND METHODS

Microencapsulation

PC12 cells (American Type Culture Collection [ATCC],Manassas, VA) were encapsulated at a density of 5 3 106

cells/mL, using a submerged jet coextrusion interfacial pre-cipitation process as described previously.9 Cells were main-tained in RPMI 1640 medium (with HEPES buffer and L-glutamine; Gibco-BRL, Grand Island, NY) supplementedwith 10% (v/v) heat-inactivated horse serum (Sigma, St.Louis, MO), 5% (v/v) fetal bovine serum (Sigma), and 1%antibiotics (penicillin [10,000 units/mL] and streptomycin[10,000 mg/mL] Gibco-BRL) and incubated at 37°C with5% CO2 in air. They were suspended in a 15% (w/v) Ficoll400 solution in serum- and antibiotic-supplementedmedium. The cells were microencapsulated in hydrox-yethyl methacrylate–methyl methacrylate (HEMA–MMA,75:25 mol%). After encapsulation, microcapsules weremaintained in supplemented RPMI and incubated at 37°Cwith 5% CO2, until implantation or analysis. Triethyleneglycol (TEG; Sigma Aldrich, Milwaukee, WI) or poly-ethylene glycol 200 (PEG, MW 200; BDH Chemicals,Poole, UK) was used as the solvent, at concentrations be-tween 9 and 12% (w/v). For capsules prepared with a pore-forming agent, poly(vinyl pyrrolidone) (PVP, MW 10,000;Sigma Aldrich) was added to the polymer solution at aconcentration of 30% (PVP/HEMA–MMA, w/w).

After 2–3 days within the supplemented RPMI, the en-capsulated cells were sorted and counted under a lightmicroscope. Capsules of acceptable quality were trans-ferred to a new petri dish with fresh medium; capsulesthat were ruptured, misshapen, or had a visibility thinwall were discarded.

Capsule and cell characterization

The number of cells per microcapsule was determinedby breaking the capsules as described previously7 andcounting the released cells in a hemocytometer with try-

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pan blue staining. The metabolic activity of the encap-sulated cells was determined by MIT assay.10

Capsule structure was assessed by scanning electronmicroscopy (SEM). Microcapsules kept in phosphate-buffered saline (PBS) were washed three times for 5 minin distilled water, cut in half with a scalpel blade, im-mersed in liquid nitrogen, and freeze dried (Labconco,Kansas City, MO) for 1–2 days. The capsules weremounted on aluminum SEM stands and sputter coatedwith gold. Microcapsules prepared in this manner wereexamined and photographed with an S-520 SEM (Hi-tachi, Tokyo, Japan).

Agarose gels

Microencapsulated cells were embedded in a disk ofagarose gel (SeaPlaque low gelling temperature agarose,type VIIA; Sigma) in order to facilitate their implanta-tion and recovery from rats. A similar protocol was usedfor intraperitoneal implants in mice.7 The agarose solu-tions were prepared under sterile conditions in a laminarflow hood. The agarose powder was dissolved in PBS at2–6% (w/v) with heat. The sol was transferred to a ster-ile scintillation vial and allowed to cool to 37°C in an in-cubator.

A mold for preparing the gels (approximate diameter,7 mm; height, 2–3 mm) was made with Silastic sheet(Dow Corning, Midland, MI); these were autoclaved andplaced into a sterile tissue culture dish before use. Be-tween 150 and 200 microcapsules were transferred bysterile pipette into the mold and the transfer PBS was re-moved. Warm agarose sol (,37°C) was dropped into themold from a 1-mL syringe, and the capsules were dis-persed throughout the agarose sol by gentle stirring witha fire-polished glass pipette tip. Gels were allowed toform at 4°C before being removed from the Silastic moldsand transferred into sterile PBS. Gel-embedded capsulesthat were kept in medium for in vitro comparison werekept in PBS for the same amount of time as were the invivo capsules before implantation.

Implantation

Microcapsules containing PC12 cells were implantedinto the peritoneal cavity or sutured omentum of rats,either by direct implantation or by first embedding thecapsules in an agarose gel. The experimental protocolwas approved by the University of Toronto (Toronto,ON, Canada) Animal Care Committee. Rats were kepton a 12 h:12 h light–dark schedule, with food and wa-ter available ad libitum. Microcapsules were kept incu-bated in culture medium for 1–2 weeks after encapsu-lation, to allow for cell growth within the capsules.Samples of microcapsules from the same batch weremaintained in vitro for the duration of the in vivo im-plant studies.

VIABILITY OF HEMA–MMA-MICROENCAPSULATED PC12 CELLS

Male Wistar rats (200–300 g; Charles River Canada,Saint Constant, PQ, Canada) inhalation of enflurane(Ethrane USP) for the duration of the surgical procedure(between 20 and 45 min). Two rats were used for eachtime point/experimental condition. For intraperitonealimplantation, 150–200 capsules in PBS were transferredby sterile transfer pipette directly into the peritoneal cav-ity through a 1- to 2-cm incision. For implantation into theomentum, an omental “pouch” was created5 by folding theomental tissue with 7-0 silk suture thread (black braided,laser-drilled needle, taper BV-1, Ethalloy; Ethicon, John-son & Johnson Medical Products, Markham, ON, Canada)and placing the capsules (with or without the agarose,same number of capsules) within. Once the capsules werein place, the muscle layer was closed with 6-0 silk su-tures (black braided, TE-1; Sherwood-Davis & Geck, St.Louis, MO) and the skin layer was closed with 4-0 silksutures (cardiopoint, black braided, CV-301; Sherwood-Davis & Geck). The wound closure was then coated withflexible collodion (Mallinckrodt, U.S.P. Un 2059;Mallinckrodt Baker, Phillipsburg, NJ). After recovery,the rats were returned to their housing and monitored forup to 1 month after surgery.

Explanation and histology

Capsules were retrieved 3, 10, or 21 days after im-plantation, by excising the omentum or by lifting the free-floating capsules from the peritoneal cavity. Explantedtissue and capsules were transported back to the tissueculture laboratory in PBS, transferred into culturemedium, and examined promptly. For the agarose gel im-plants, the gels were separated from the surroundingomental tissue with surgical tweezers and a scalpel blade.The capsules were then gently separated from the agarosewith a scalpel blade and needle, and incubated in culturemedium.

Some capsules were set aside for MTT assay and somefor cell counts. Selected capsules and explanted tissuewere rinsed with fresh PBS and fixed for histology byplacing them into a glass scintillation vial with 10 mL of 10% formalin (1 part 37% formaldehyde [SigmaAldrich], 9 parts PBS). Fixed samples were kept refrig-erated until sectioning. Fixed samples were washed twicefor 15 min with PBS and cryoprotected with 30% (w/v)sucrose in PBS overnight at 4°C. Samples were frozen-embedded in O.C.T. embedding compound (SakuraFinetek U.S.A., Torrance, CA) and stored on dry ice ina plastic mold (Sakura Finetek U.S.A.). Five- to 10-mMcryostat sections were cut at 220°C and mounted ongelatin–chrome alum-treated slides (0.1% [w/v] gelatin,0.01% [w/v] chromic potassium sulfate). O.C.T. com-pound was washed from the sections with water. Sectionswere stained either with aqueous toluidine blue (0.1%[w/v] in distilled water; BDH Chemicals), Masson

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trichrome (Sigma), or Harris hemotoxylin and alcoholiceosin (Sigma). Histological sections were viewed with an Axiovert 135 inverted epifluorescence microscope(Zeiss, Oberkochen, Germany).

RESULTS

Capsule structure

Capsules with an average diameter of about 400 mmwere prepared under the conditions used here. The cap-sules were not well centered, however: the wall thicknessvaried from 30 to 150 mM (Fig. 1). The SEM imagesshow a thin skin layer of cured polymer on both the in-tra- and extracapsular side of the membrane; the regionwithin the membrane appears to be composed of eitherfinger-like macrovoids or a porous, spongy polymer re-gion. The general structure of the two capsule membranesis similar, but the macrovoids appear to be more signif-icant in the 9% HEMA–MMA/TEG polymer preparedwith PVP.

Microcapsule implantation without agarose

Microencapsulated PC12 cells were used as an allo-geneic transplant model and were directly implanted intothe peritoneal cavity and the omental pouch of male Wis-

FLEMING AND SEFTON

tar rats. There was no intention of exploiting the dopa-mine-secreting characteristics of the PC12 cells in thismodel. Capsule implantation into a presutured omentalpouch required approximately 30–40 min per rat; directintraperitoneal implantations were completed in 10–15min each. First, the omental tissue was folded and su-tured; the capsules were then transferred from PBS intothis pouch. The omental tissue was stretched and suturedagain to enclose the capsules and prevent them from“falling out” of the pouch. The omentum was then shiftedaway from the opening to prevent adhesion to the woundsite. Capsules could usually be seen within the omentalpouch through the thin fascia-like tissue at the time ofimplantation.

Microcapsules were implanted into rats for a periodranging from 3 days to 3 weeks. No obvious changes inthe behavior or activity of the rats were observed duringthis time and they gained weight at normal rates. For theintraperitoneal implants, less than 10% of the original im-planted capsules were found free-floating in the peri-toneal cavity. Most capsules were found loosely attachedto the intestines, muscle wall, stomach, or liver. Theseattached capsules, which were gently removed withtweezers or a surgical blade, accounted for 20–60% ofthe original implanted capsules. The remainder of thecapsules could not be found. A visual search was ham-pered by the size and semitransparency of the capsules

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FIG. 1. SEM of capsules prepared with two of the polymer solutions studied here: (a) 10% HEMA–MMA in TEG and (b) 9%HEMA–MMA in TEG with additional PVP.

and the many folds among the organs in the abdomen.Recovered capsules were placed directly into PBS.

The omental pouch procedure provided a simple andefficient recovery procedure. The abdominal cavity wasexposed, and the omental pouch was excised and placedinto PBS. With approximately one-quarter of the rats, theomentum was found adhered to the liver, spleen, or stom-ach; this required careful separation from the tissue so asto minimize the transfer of blood or intestinal contentswith the omental pouch removal. The peritoneal cavitywas then examined for capsules that had fallen out of thepouch, which were also transferred to PBS (generally notmore than 5–10 capsules were found outside of the su-tured omental pouch).

A large percentage of the capsules in the histologicalsections appeared damaged or misshapen. Toluidine blueaqueous staining allowed for staining of the cells with-out dissolving the capsule wall, and in most cases thisshowed microcapsules that were no longer intact (Fig. 2).Capsules recovered from intraperitoneal implants alsohad a “flattened” appearance or were otherwise damaged.As well, many of the recovered capsules were enclosedin a fibrous overgrowth. The microcapsules could be sep-arated from this surrounding tissue with a scalpel blade,but even then a layer of host cells on the capsule surface(about five cells thick) could be seen under a microscope.Staining with MTT suggested that only a few of the en-capsulated cells remained viable after intraperitoneal im-plantation (not shown).

VIABILITY OF HEMA–MMA-MICROENCAPSULATED PC12 CELLS

Attempts to physically separate intact capsules fromthe omental pouch tissue were not successful. This led toincubating the explanted omental pouch in a collagenoussolution, which served to dissociate most of the sur-rounding tissue and free some of the capsules. Manipu-lation of the remaining tissue with a scalpel allowed forthe isolation of more capsules, although recovery neverexceeded 30% of the original implant number. Typically,a large capsule aggregate was isolated. It appeared to beboth compacted together and enclosed in a fibrous hosttissue response, suggesting that the capsules were beingflattened or ruptured in vivo. As before, the recoveredcapsules were separated from the surrounding fibrous tis-sue and analyzed, but no viable cells were found.

Microcapsule implantation with agarose

Initial studies were conducted with soft ultra low-gelling agarose (5%), but this agarose was too “degrad-able” for in vivo use: it had disappeared within 3 weeks,with some loss of material apparent at 10 days.11 Ratherthan increasing the agarose concentration, we chose towork with SeaPlaque low-gelling agarose. The low-gelling agarose gel reverts to a sol at 55–60°C, but thegel forms between 27 and 30°C. This temperature rangepresents a slightly more difficult handling issue whenworking with live cells, but still lies within tolerable lim-its. A concentration of 4% (w/v) agarose (in PBS) waschosen. At this concentration, the low-gelling agarose

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FIG. 2. Toluidine blue-stained microcapsules (without agarose) within an omental pouch after 3 weeks of implantation. Cap-sules are no longer spherical and appear to be crushed and broken (see arrows). No viable encapsulated cells are apparent in thissection. Capsules have also agglomerated within the omental tissue. Scale can be inferred from 400-mm capsule diameter.

gels are approximately three times stiffer than the 5% ul-tra low-gelling agarose (according to the supplier infor-mation). The effect of the agarose on cell viability (MTTassay) is shown in Fig. 3 for capsules incubated in vitro,while embedded in agarose. A negative impact on cellproliferation (MTT activity at later times) appears statis-tically significant with the 10% HEMA–MMA/TEG cap-sules, but not with the 9% HEMA–MMA/TEG plus PVPcapsules. Conceivably this may be due to a lower MTTdiffusivity in the less permeable capsules, but our expe-rience with MTT assays and HEMA–MMA capsules hasnever hitherto supported such an explanation.

The implantation procedure was simplified and im-proved with the low-gelling agarose; the gel could be eas-ily lifted and placed into the sutured omentum withoutrisk of breaking the gel (unlike the ultralow-gellingagarose) and there was no need to transfer (and remove)excess PBS (as with capsules alone). The gel was heldfirmly in place with minimal suturing, and the gel withcapsules inside could be seen through regions of fascia-like tissue pulled across the gel. At the end of the im-

FLEMING AND SEFTON

plantation period, the omental pouch usually had a red-dish-pink appearance, with little inflammation of theomental tissue. The low-gelling agarose gel was visiblethrough thin regions of omental tissue, over which spreada considerable amount of vasculature. These branchingblood vessels, which were not seen before implantation,were consistently present in the thin tissue proximal tothe gel. The low-gelling agarose gels were intact on ex-plantation (Fig. 4), with no noticeable changes in size,shape, or consistency after the three weeks. The intactagarose gels could be easily ‘squeezed’ out of the ex-planted omental pouch, which left behind a thin pocketof vascularized fibrous tissue. There appeared to be lit-tle damage to the surrounding tissue upon removal of theagarose. A degree of host cell infiltration could be seenon the outside layer of the gel (Fig. 4). This increasedover the three week period, up to a depth of approxi-mately 100 mm into the gel. As well, capsules locatedclose to the edge of the gel had a layer of cells on theirsurface (Fig. 4b). However, both the capsules and gel po-sitioned away from the periphery were free of host cells.

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FIG. 3. MTT activity of microencapsulated PC12 cells embedded in 4% low-gelling SeaPlaque agarose in comparison withcapsules not in agarose. In vitro studies: (a) 10% HEMA–MMA in TEG capsules; (b) 9% HEMA–MMA in TEG with 30% PVP(w/w with HEMA–MMA). Agarose reduced MTT activity at longer times, but the effect was significant only for 10%HEMA–MMA in TEG capsules. (Means 6 SD, n 5 3, 10 capsules per well.)

Microcapsules after recovery

The capsules were gently removed from the gel; thereappeared to be no adhesion. As much as 90% of the orig-inally implanted capsules were retrieved from the low-gelling agarose gels. Capsules that remained intact andspherical were selected for MTT and cell count assays;approximately 10% of the capsules were damaged or miss-hapen. A significant loss of cell number and viability wasseen over the 3 weeks of implantation with both types ofcapsules (Fig. 5). Cell viability (MTT) in vivo was much

VIABILITY OF HEMA–MMA-MICROENCAPSULATED PC12 CELLS

less after 3 weeks than was obtained in vitro. However,no difference between in vivo and in vitro viability re-sults was seen after 3 days of implantation, and after 3weeks the number of viable cells was about half that onimplantation: in previous studies without agarose, no vi-ability had been seen after 1 week. The MTT assay re-sults are significantly higher for capsules with cells thanfor explanted blank capsules, even after 3 weeks in vivo.Explanted capsules were also incubated with MTT solu-tion to confirm the presence of viable cell aggregateswithin an intact polymer membrane (data not shown).

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FIG. 4. Light micrographs of (a) agarose disk and (b) microcapsules after implantation in an omental pouch for 3 weeks. The4% low-gelling agarose disk was cleanly separated from the omental tissue. The latter was not adherent to the agarose and thecapsules inside the gel were intact and maintained their sphericity. In (b) capsules separated from the periphery of the gel havea layer or two of host cells surrounding them. On the other hand, a capsule removed from the middle of the gel (arrow) wasclean. Scale can be inferred from 400-mm capsule diameter.

The loss of viability and cell number appears to be greaterwith the 9% HEMA–MMA/TEG plus PVP capsules, al-though it is difficult to ascribe much significance to thedifference between the two capsule formulations.

Histology (Fig. 6) showed that after 3 weeks in vivo,the agarose gel appeared to be surrounded by a layer ofactive, mature fibroblasts. Next to this seemed to be alayer of elongated fibroblasts and dense connective tis-sue, with some collagen fibers running parallel to the gelsurface. There appeared to be more collagen present whencapsules were embedded in the agarose gel, comparedwith agarose disks alone (data not shown). Next was alayer of irregular and loose connective tissue, with nu-merous blood vessels present. Further away from the gel,there were layers of adipose connective tissue, with col-lagen and active fibroblasts; inflammatory cells also ap-pear (neutrophils, basophils, lymphocytes, and mono-cytes). The cell aggregates within the capsules generallyappeared quite healthy; 20–40% of the cells (those to-ward the center of the aggregates) were pale with smalldark nuclei, whereas the darker cells closer to the pe-

FLEMING AND SEFTON

rimeter of the aggregate had smooth cell membranes andlarge round nuclei. Some of the capsules positioned nearthe periphery of the gel had large, poorly defined cellson the outside of the capsules; some were multinucleate,possibly polymorphonuclear neutrophils or basophils.Some also had an undetermined fibrous component ontheir surface, possibly collagen.

DISCUSSION

This study had two objectives: (1) to use agarose disksas a means of enhancing encapsulated cell viability invivo (and of enhancing localization and recovery at ex-plantation) and (2) to compare two formulations of mi-crocapsules with a view to determining which was prefer-able.

Agarose disk implantation

From a surgical point of view, suturing the omentuminto a pouch provided an excellent method of localizing

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FIG. 5. MTT assay and cell counts of microcapsules after recovery from 4% low-gelling agarose disks, 3, 10, and 21 days af-ter implantation in comparison with gel-embedded capsules maintained in vitro. (a and b) 10% HEMA–MMA capsules in TEG;(c and d) 9% HEMA–MMA capsules in TEG with 30% PVP. (a and c) MTT assay; means 6 SD, n 5 3, 10 capsules per well.(b and d) Number of cells per capsule 6 5%. In vivo results are the average of two rats at each time point; in vitro results areidentical to those shown in Fig. 3.

and retrieving microcapsules. Containment in one areaminimized the different types of tissue that the capsulescame into contact with, allowing for a more consistentand reliable analysis of the host response to transplantedcapsules than peritoneal cavity implantation provides.5

Clinical application of microencapsulation will likely ne-cessitate complete retrievability, which could be obtainedby surrounding the capsules with a removable enclo-sure—the omentum satisfies this requirement. The omen-tum is richly vascularized,12 making it additionally use-ful as a target site for our purposes. However, directlyimplanting the capsules into an omental pouch (withoutagarose) was not useful because many of the capsules ag-glomerated together and became misshapen. This causeda serious diffusion limitation for the capsules situated to-ward the center of the clump. Also, capsules that weretightly packed together were presumed to have a highchance of rupturing and releasing the cells inside. Suchclumping was noted by others7 using different cells, an-imals, and implantation sites, leading to the use of anagarose disk as an embedding medium before intraperi-toneal implantation. This study confirms the results ob-tained earlier7 with a different animal and encapsulatedcell model (rat versus mouse; PC12 versus L929); eventhe extent of viability retention after 21 days (,50%) wassimilar.

VIABILITY OF HEMA–MMA-MICROENCAPSULATED PC12 CELLS

The implantation protocol was greatly eased by em-bedding them within agarose gels. Capsules were im-planted all at once, without risking capsule loss or dam-age during transfer. The time required for omental pouchsuturing was also considerably decreased, reducing theamount of time spent under anesthetic. On explantation,capsule retrieval was close to 100%, without having tosubject the capsules to the physical or enzymatic stressesinvolved with separating them from tissue. The “bufferzone” provided by the agarose gel between capsules andthe host tissue may also minimize the inflammatory re-sponse to the capsules. One concern is with proteins ad-sorbed onto the microcapsule surface from the incuba-tion medium. Although capsules were rinsed with PBSbefore implantation, previous studies6 have found that ad-sorbed proteins remain on the surface even after wash-ing with PBS. In other studies, capsules were incubatedin serum/protein-free medium before implantation tominimize this problem. The agarose also disperses pres-sure forces in the omental pouch and provides structuralsupport for the capsules, which prevented them from be-ing crushed or broken.

Agarose has been used for immunoisolation studies,13,14

but its lack of selectivity limits its utility. Here the per-meability selectivity is provided by the HEMA–MMAcapsules and the high permeability of agarose was a de-

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FIG. 5. (continued)

sirable attribute to maximize nutrient transport. Most ofthe work with agarose immunoisolation devices involveda similar low-gelling temperature agarose, as used here.This gives a high gel strength with a melting tempera-ture .60°C. The gel formation temperature, about 30°C,is still suitable for working with live cells. Degradationstudies have reported a 20% decrease in gel strength forthe low-gelling agarose over 90 days at 37°C,15 whichshould still provide sufficient strength for the applica-

FLEMING AND SEFTON

tions presented here. The manufacturer reported thatlow-gelling agarose gels were more than three timesstronger than ultralow-gelling agarose at the same con-centrations.

The long-term stability of agarose within the omentalpouch was not explored in the 3-week studies presentedhere. The consequence and likelihood of agarose degra-dation in vivo would have to be defined before its use forembedding capsules became standard practice. Agarose

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FIG. 6. Histological sections of omental tissue surrounding explanted agarose gel containing 10% HEMA–MMA in TEG cap-sules on day 10. (a) Darkly stained viable cell aggregate (arrow) is visible at lower left; capsule dissolved during H&E staining.(b) Higher magnification view of agarose–omentum interface from a different section. Fibroblasts shown adjacent to agarose onthe left side of image. Scale can be inferred as above.

gels may serve their purpose of isolating and protectingthe capsules for the first few weeks; this may allowenough time for the immune/inflammatory response to“settle down.” The eventual disappearance of the em-bedding device may even be a target with future studies,so that the diffusion and response times are gradually in-creased. No literature was found indicating agarose atthese concentrations to be unstable in vivo; indeed, 5%agarose microbeads were suggested to be stable for atleast several hundred days in rodents.14,16

Biocompatibility was a concern with the use ofagarose. Although agarose is reported to be nontoxic invivo,14,17 some degree of inflammatory response was ex-pected given the nature of the accompanying surgery.However, the histological sections of the omental tissuesurrounding agarose implants (Fig. 6) showed an ac-ceptable host response to the gel. A thin tissue layer im-mediately next to the gel consisted mainly of fibroblastsand collagen. There was little macrophage and neutrophilinfiltration and this indicated a degree of tolerance to thegel. The presence of blood vessels approximately 10 celllayers into the tissue is encouraging, as they indicate anadequate source of nutrients and oxygen for the trans-planted cells. A similar acceptable host response wasnoted for agarose microbeads implanted in the peritonealcavity.18

Subsequent studies have highlighted the importance ofminimally invasive procedures to reduce the inflamma-tory response associated with microcapsule implanta-tion.19 Even sham surgeries, without capsule implanta-tion, as used for intraperitoneal implantations (let aloneomental pouch formation), were found to be sufficient toactivate the peritoneal macrophages to a high extent. Thisprecluded delineation of the more subtle effects associ-ated with capsule structure or wall chemistry. In this min-imally invasive protocol capsules are suspended in warmagarose before injection through a catheter to realize thebenefits of agarose embedding but without the negativeconsequences of macrophage activation.

Encapsulated cell viability. An advantage to usingagarose gels for implantation is that the explanted cap-sules can be quantitatively analyzed by the same meth-ods used in vitro. Previous studies have relied on histo-logical sectioning for an assessment of explanted cellviability; MTT and cell count assays require capsules tobe intact and free of tissue, and therefore could not beused for comparison.

Embedding capsules in agarose had a small but mea-surable effect on cell proliferation inside the capsuleswhen the disks were incubated in vitro. This differencewas more significant with the 10% HEMA–MMA/TEGcapsules than with the 9% HEMA–MMA/TEG plus PVPcapsules, which were thought to be more permeable (Fig.3). It is possible that the encapsulated cells were affected

VIABILITY OF HEMA–MMA-MICROENCAPSULATED PC12 CELLS

by the preparation of the agarose gels. The capsules aretransferred to PBS for 10 min, exposed to warm agarosesol, and kept in the refrigerator for the gels to set. Thecapsules are then kept in PBS for up to 1 h before re-ceiving nutrients again. The growth curves appear to havea “lag” in growth; for the 9% TEG/PVP capsules, afterthe first 7 days the growth rate appears to be indistin-guishable from that of nonembedded capsules.

Both cell proliferation and viability after 3 days in vivowere comparable to in vitro results; perhaps this is dueto the residual medium in the capsule core on implanta-tion. By 21 days viable cell number and MTT results wereconsiderably less than those at day 3 (counting from thetime of implantation) and much less than that in similarcapsules maintained in vitro for the same time period(counting from the day of encapsulation). Nonetheless,there were some viable cells (and MTT activity) presentin the capsules at 21 days: about half the number that wasthere at day 0 of implantation. After 21 days, histologi-cal sections revealed that cells at the aggregate core werenecrotic, whereas viable cells were seen at the periphery.This is indicative of diffusion-limited spheroidal growth;host rejection of the cells would probably indicate the re-verse, with greater necrosis at the periphery. The pres-ence of some viable cells is significantly different fromearlier studies with allogeneic (and xenogeneic) livercells in rats, where no viable cells were found by histol-ogy 14 days after implantation of HEMA–MMA cap-sules.5 That study did not use agarose gel as a preim-plantation embedding medium. When agarose was usedin a syngeneic model (L929 cells in C3H mice),HEMA–MMA-encapsulated cells survived for 21 days,consistent with the results shown here.

The diffusion limitations introduced by the agarosegels are of a concern if they impact cell growth. For PC12cells, the differences are barely significant but this issuemay be more important for more sensitive cells. Therehave been several attempts to characterize the diffusionproperties of agarose gels. Li et al. investigated 4%SeaPlaque (low-gelling) agarose, determining that thegels offered little transport resistance for solutes up to150 kDa.20 Oxygen diffusivities were found to be about95% of those for pure water, and a diffusion coefficientfor ovalbumin (43.5 kDa, comparable to horseradish per-oxidase, HRP) was on the order of 4.4 3 1027 cm2/s.Westrin et al. also presented data for 4% agarose beads(1-mm diameter); diffusion coefficients for medium-sized species were on the order of 8 3 1026 cm2/s.21

Yang et al. investigated small molecule diffusion in 5%agarose gels, reporting diffusion coefficients of about5 3 1026 cm2/s for both glucose and insulin, close totheir diffusion in water.16 Iwata and coworkers, who haveworked extensively with agarose microbeads, reported analbumin (,60 kDa) diffusion coefficient on the order of1 3 1027 cm2/s.18

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These results consistently indicate that diffusion resis-tance through agarose is at least an order of magnitudelower than through HEMA–MMA capsules. Previous de-termination of HEMA–MMA capsule permeability indi-cated that the microcapsules had a diffusion coefficientfor HRP (40 kDa) on the order of 1029 cm2/s; with themost permeable capsules yielding a measurement ofabout 3 3 1028 cm2/s. However, there are two additionalfactors to consider. A diffusion coefficient for a 40-kDaprotein in 4% agarose is estimated to be 4 3 1027 cm2/s;for the same molecule through HEMA–MMA the esti-mate is 4 3 1028 cm2/s. Although the capsule coefficientis lower, the distance traveled is higher in the agarose.Capsule walls were about 100 mm thick, whereas the dis-tance to the center of the agarose gels implanted in thisstudy was approximately 1 mm. Thus the two diffusionresistances are comparable, with the result that the pres-ence of agarose likely doubles the diffusion resistancecompared with capsules alone. If these capsules are morepermeable than those studied previously, then the nega-tive effect of agarose should be much greater. Obviously,this is a concern for diffusion-limited cell growth, andprobably contributes to the loss of cell viability seen inthis study.

Effect of capsule formulation. Previous experiments8

showed that a significant increase in permeability tohorseradish peroxidase (HRP) can be achieved by de-creasing the concentration of HEMA–MMA in the sol-vent (TEG), and/or by introducing PVP to the polymersolution as a “pore-forming agent.” These findings werethe motivation behind comparing the two main polymersystems used for this study, 10% HEMA–MMA/TEG and9% HEMA–MMA/TEG plus PVP. On the basis of pre-vious results, capsules prepared with these two materialsshould differ in permeability to HRP by approximatelytwo orders of magnitude. One goal of this study was todetermine whether this permeability difference wouldhave a significant impact on cell growth or viability thatcould be seen either in vitro or in vivo. The capsules pre-pared with PVP also had some minor advantages: betteroverall capsule quality, less tackiness in vitro, and opac-ity (for easier explantation).

Membrane structure was similar between the two cap-sule types; the dense spongy layer was more distinctivefor the 10% HEMA–MMA/TEG capsule wall, whereasthe macrovoid spaces are more apparent with 9%HEMA–MMA/TEG plus PVP. Although core definitionwas good, with little internal mixing between core andpolymer, there was also a significant degree of core ec-centricity as is inherent to capsule formation, even withthe “highest quality” capsules. For both formulations, thecapsule permeability to HRP was much higher than pre-vious batches8: here most of the loaded HRP was releasedwithin the first 30 min, precluding quantitative measure-

FLEMING AND SEFTON

ment and comparison. Previously, it would take .8 h torelease 20% of the loaded HRP in the less permeable 10%HEMA–MMA/TEG capsules.8 There is no clear reasonfor the higher permeability, although differences in op-erator and in polymer batch (molecular weight, branch-ing) are likely causes.

Consistent with the expected differences in perme-ability, there was a small increase in PC12 cell growthrate (based on MTT; Fig. 3) for the “more permeable”9% HEMA–MMA/TEG plus PVP microcapsules. PC12cells proliferated consistently in both capsule types overseveral weeks, with no significant loss of viability in ei-ther capsule (in vitro). In vivo, the loss of viability wassimilar for both cases and it is not possible to make acomparison between the two formulations on the basis ofthe in vivo data. Encapsulated PC12 growth was slowerthan growth in conventional two-dimensional (2-D) cul-ture, as was noted previously.22 The effect of capsule for-mation on PC12 growth was small; the effect for othercell types was more significant. In other studies, after 4weeks in culture, L929 cells (a mouse fibroblast line)grew inside 9% HEMA–MMA/TEG plus PVP capsulesto have an increase in MTT signal (from day 0) that wasfive times larger than the increase obtained for 10%/TEGcapsules.12 Unfortunately, the presence of only (small)qualitative differences in structure and in vitro cellgrowth precludes unequivocal discussion of cell perfor-mance in terms of capsule permeability.

Need for more permeable microcapsules

There are many factors that favor capsules of higherpermeability. The capsule membrane would be expectedto limit diffusion and decrease cell doubling time. Thediffusion situation only becomes worse in vivo, with cap-sule aggregation, poor nutrient availability, fibrous re-sponse, and low O2 partial pressures being introduced.Using agarose adds extra diffusion limitations, makingan increase in capsule wall permeability desirable fortransplanted cell viability. However, the use of agarosegel helps to alleviate the mechanical stress on capsulesin vivo, enabling the use of thinner, otherwise mechani-cally weaker capsules. An optimal level between the me-chanical and diffusive properties for each capsule and/orembedding device system needs to be found.

If more permeable capsules become an objective forHEMA–MMA microencapsulation, the ability to consis-tently attain perfect core centering would allow capsulesto be made with thinner membranes and sufficient phys-ical strength. The current encapsulation needle assemblyis a prototype suitable for experimental use; future de-sign improvements would require some means of ensur-ing that the inner core needle remains perfectly centeredwithin the polymer needle. Improvements to the repro-ducibility of the encapsulation process would also be re-

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quired for the consistent production of high-quality cap-sules. The current percentage of acceptable quality cap-sules is too low for larger scale production, and the sort-ing of a batch of microcapsules under a light microscopehas its limitations.

CONCLUSIONS

Embedding HEMA–MMA microcapsules within gelsprepared with 4% low-gelling agarose improved the en-tire omental pouch implantation procedure. These gel de-vices were easier to implant and retrieve than microcap-sules alone, and protected the microcapsules from fibrousovergrowth and damage due to pressure or capsule ag-gregation. On explantation, the intact and spherical mi-crocapsules could be assessed with the same methodsused to analyze capsules maintained in vitro. Agarosebiocompatibility and durability were found to be accept-able, at least for the purpose and duration needed here.

The use of agarose also improved in vivo-encapsulatedcell viability, when compared with previous implantationstudies. PC12 cells implanted with agarose proliferatedin vivo during the few days of implantation. Cell viabil-ity decreased over the 3-week implantation period, butapproximately 50% of the initial cells implanted werestill viable at 21 days. This is a significant improvementover previous work, which reported no cell viability forliver cell allografts past 7 days in vivo, also in an omen-tal pouch.

PC12 cells grew in vitro within both 10% HEMA–MMA/TEG and 9% HEMA–MMA/TEG plus PVP mi-crocapsules, albeit at a slower rate than in conventionaltissue culture. PC12 cells appeared to proliferate fasterin the 9% HEMA–MMA/TEG plus PVP capsules (invitro), consistent with the expected higher permeabil-ity. However, the permeability for both capsule typeswas higher than expected, so as unequivocal relation-ship between cell viability and permeability is not pos-sible.

ACKNOWLEDGMENTS

The authors acknowledge the financial support of theMedical Research Council (now Canadian Institutes ofHealth Research) and the Natural Sciences and Engi-neering Research Council. We also acknowledge thetechnical support of Chuen Lo and Dagmar Gross.

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VIABILITY OF HEMA–MMA-MICROENCAPSULATED PC12 CELLS

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Address reprint requests to:M.V. Sefton, Ph.D.

Department of Chemical Engineeringand Applied Chemistry

University of TorontoToronto, ON M5S 3G9, Canada

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