Microencapsulation of viable hepatocytes in HEMA-MMA microcapsules: a preliminary study

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Mi~~e~caps~latio~ of viable hepatocytes in HEMA-MMA microcapsules: a preliminary study G.D.M. Wells*, M.M. Fisher+ and MY, Sefton*

*Department of Chemical Engineering and Applied Chemistry and Centre for Biomaterials Hospital, University of Toronto, Toronto, Ontario, M5S 1A4, Canada

and tSunnybrook

Viable rat hepatocytes were encapsulated in a HEMA-MMA copolymer (80% HEMA). Encapsulated hepatocytes continued to produce urea (a measure of viabili~) for approximately 2 wk although urea production rates fell steadily over the course of in vifro culture in a pattern similar to those of control hepatocytes in conventional culture. Urea production was slightly higher in 0.01 M Tris buffered glycerol precipitated capsules, relative to phosphate buffered saline precipitated capsules. Hepatocytes were not viable in 0.001 M Tris buffered glycerol precipitated capsules which had a dense wall without the macroporosity seen in the walls of the other capsules. More work is needed to show that HEMA-MMA encapsulated hepatocytes retain some of the differentiated functions of hepatocytes. Keywords:

Microencapsulation,

hepatocytes,

HEMA-MMA,

urea

Received 10 June 1992; revised 28 December 1992; accepted 30 December 1992

An ‘artificial liver’ has been proposed as a means of replacing liver function in cases of acute or chronic hepatic failure. In the case of acute fulminant hepatic failure temporary liver support might allow the patient to survive until the liver can regenerate. In chronic failure, the ‘artificial liver’ could be effective as a bridge to liver transplantation or even as an alternative to transplantation. Haemoperfusion with various adsorbents’, plasmapheresis’ and immobilized enzymes3 are among the techniques used clinically or at least proposed for temporal, partial liver support. Hepatocytes cultured on artificial capillaries4, or microcarrier@ or dialysis against viable hepatocytes’ have also been considered. For example, prolonged survival and function of transplanted hepatocytes (as much as >180 d) have been shown using a biodegradable support of high surface area7 or using Goretex fibres coated with collagen and heparin binding growth factor’. These special structures were intended to promote neovascularization of the implant to provide the vascular support for the transplanted cells. Immune suppression or isolation would still, however, be necessary. Mi~~capsulation, like other immunoisolation methods, is being considered as a means of avoiding immune suppression in the use of hepatocytes. Diffusion of O,, nutrients and metabolites would be allowed to sustain the cells surrounded by a thin spherical shell consisting of a biocompatible semipermeable polymeric membrane. Substrates requiring detoxification or low molecular Correspondence to Dr M.V. Sefton. 0 1993 Butterworth-Heinemann 0142-9612/93/080615-08

Ltd

weight protein products of the cells also diffuse across the membrane. However, the membrane is impermeable to antibodies and lymphocytes so that immune suppression would not be needed. Furthermore it may be possible to use xenogenic hepatocytes obviating the problems of limited donor availability. Viable hepatocytes were encapsulated’ in the alginate-polylysine systems used to encapsulate pancreatic islets. Albumin, in addition to 10 other proteins, was released from the encapsulated hepatocytes”. In rats with galactos~ine-induced fulminant hepatic failure, more than 50% of the encapsulated hepatocytes were viable 35 d after implantation (ip). Others report similar benefits of alginate-polylysine encapsulated hepatocytes in terms of increased survival in models of fulminant hepatic failure”, I2 or in lowering bilirubin levels13. Here we use a hydroxyethyl metha~~late-methyl methacrylate copolymer (HEMA-MMA) that is potentially more biocompatible than alginate-polylysine. Intraperitoneal implantation in rats of these capsules (without cells) resulted in only a very thin fibrous capsule (- 5 pm thick) after 4 wk, unlike the much thicker foreign body reaction seen with alginate-polylysine. Capsules similar to those prepared here have a molecular weight cut off of 100 00014, sufficient to exclude immunoglobulins from the encapsulated cells. HEMA-MMA capsules are also tough mechanically, unlike the more fragile alginate capsules. Unfortunately the polymer is not water soluble, necessitating the use of an organic solvent (polyethylene glycol 200) in encapsulation: this makes encapsulation more difficult (compared with the water soluble alginateBiomaterials

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polylysine). The water insolubility is, however, considered an advantage since this is presumed to be a key factor leading to its greater biocompatibility. A variety of viable cells have been encapsulated in HEMA-MMA, including pancreatic islets15, Chinese hamster ovary (CHO) cells’6-‘8, dopamine producing cells (PCZ~*~) and more recently liver tumour cells (HepG2”). With the exception of islets, all have been relatively robust cell lines. Primary hepatocytes are different in that they can be expected to be more sensitive to encapsulation conditions and less ‘forgiving’ of process difficulties. Hepatocytes also require an extracellular matrix for support: the matrix needs to be added to the cell suspension prior to encapsulation. Matrigel@ is used with the current experiments with HepG2 cells; for these earlier studies collagen was used for hepatocytes; because collagen gels readily, this makes microencapsulation more difficult than for cells (e.g. CHO, PC12) that grow in suspension. Here, we demonstrate the in vitro viability of HEMA-MMA encapsulated hepatocytes. Consistent with the preliminary nature of this work, we used only urea production as a marker.

MATERIALS

AND

METHODS

Hepatocytes Hepatocytes ((l-4) X lo* cells/rat) were obtained by in situ collagenase perfusion of the livers of male Wistar rats (50-200 g, Charles River Laboratories, Montreal, Quebec). Animal protocols were approved by the local Animal Care Committee in compliance with the rules of the Canadian Council on Animal Care. Viabilities ranged from 85 to 90%, measured using trypan blue. Livers of anaesthetized rats were perfused via the portal vein with a calcium free buffer (g/l: 8.3 NaCl, 0.5 NaCl, 2.4 Hepes, pH 7.4) Followed by Ca2+ containing buffer (g/l: 3.9 NaCl, 0.5 KCl, 2.4 Hepes, 0.7 CaCl,, 2 H,O, pH 7.8) with 100 units/ml collagenase [Worthington Biochemical, Freehold, USA). The liver was removed and, following disruption of the surrounding Glisson’s capsule and gentle dissociation of the lobes, the cells were dispersed in RPM1 1640 (Gibco, Grand Island, USA) supplemented with fungizone, penicillin/streptomycin and a mixture of growth factors based on the defined medium of Enat et al.” plus hydrocortisone hemisuccinate (Solu-Cortef) (1 pg/ml), and linoleic acid (5 pg/ml in 0.4 g/ml bovine serum albumin in phosphate buffered saline (PBS]). Hepatocytes (3.2 X 106/5 ml) were grown on collagencoated Petri dishes. The latter were prepared by adding 1 ml of collagen (pH 7.4, Vitrogen 100, Collagen Corp., Palo Alto, USA) to each dish and allowing it to gel overnight before use. For encapsulation, the cell suspension consisted of 2 ml of -6 X lo6 cells/ml RPM1 (complete] diluted with 3 ml of 2% sodium alginate (Kelcogel LV, Chicago, USA] and 1 ml of Vitrogen 100 solution (pH 7.4). The vitrogen solution was prepared by mixing 2.4 ml of stock solution with 0.1 N NaOH (0.3 ml) and 0.3 ml of 10 X PBS. The sodium-alginate was added to the cell suspension to increase the viscosity and minimize settling during encapsulation. Biomaterials 1993, Vol.

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hepatocytes

in HEMA-MMA

microcapsules:

G. D. hf. Wells et al.

Polymer Unlike other studies’5-20 where a 75% HEMA polymer was used, here the polymer had a nominal composition of 80 mol% HEMA/ZO mol% MMA. The 80% HEMA copolymer was expected to have a higher permeability to proteins the size of albumin because of the expected higher water content; the permeability was not measured directly. The polymer was prepared by solution polymerization using azobisisobutyronitrile (0.001 mol/mol of monomers] as initiator”. The polymer solution normally consisted of 9% (w/v) HEMA-MMA in polyethylene glycol (PEG 200, Sigma, St Louis, USA). Occasionally the solvent was 85% PEG 200/15% water (v/v).

Microencapsulation The submerged jet process as described elsewhere” was used here. Hepatocytes in a collagen solution and polymer solution were pumped (0.020 ml/min, 0.047ml/ min, respectively) to a coextrusion needle which was at the end of a 9 in arm attached to a cam and motor assembly. Movement of the needle up-and-down (30 cycles/min) and hence in-and-out of a 7-8 cm layer of hexadecane in a 250 ml volumetric flask resulted in the formation of a droplet of controlled size. Droplets were formed at the rate of 15/min. The droplets fell into the magnetically stirred aqueous precipitation bath which filled the bulk of the flask. As soon as the droplet came into contact with the water, the PEG was extracted leaving the polymer behind to precipitate as a shell around the cells. The precipitation bath was either PBS 0.14 M NaCl, 8.1 mM NazHPO,, 0.98 mM KH,PO,; pH 7.4) or 0.3 M glycerol, buffered with 0.01M Tris; both contained 50 p.p.m. Pluronic LlOl (BASF, Wyandotte, USA). After 25 min in the precipitation bath, the capsules were washed in Williams E medium and placed in 5 ml of complete RPM1 1640 in a Petri dish and maintained in the incubator at 37’C, 95% sir/5% CO,.

Urea nitrogen

assay

Following the start of new cultures of hepatocytes both in the control monolayers and the microcapsules, the growth medium was changed approximately every day and the used medium was stored at -2O“C for subsequent urea analysis. Blood urea nitrogen reagents and standards (Sigma) were used in the standard fashion. Each sample and standard was run in duplicate. Results were converted into micrograms of urea nitrogen per million cells per 24 h.

Scanning

electron

microscopy

Capsules were frozen in liquid nitrogen and freeze dried (Lyph-lock 6, Labconco Inc., Kansas City, USA). A razor blade was used to fracture the freeze dried microcapsules and these were mounted on an aluminium scanning electron microscope (SEM) stud using double sided tape and coated with gold using a Polaron SEM coating system. The capsules were then examined using a Hitachi 5520 SEM.

Microencapsulation

of viable hepatocytes in HEMA-MMA -

RESULTS

Microencapsulation Submerged jet extrusion into PBS was used to prepare microcapsules with hepatocytes on a regular basis. The capsule diameter was -900 pm and the wall structure is shown in Figure 1. The wall thickness was on average -60 pm, although wall thickness occasionally varied from -20 to -150 ,um both among different capsules of the same batch and occasionally within a single individual capsule: in the latter case the capsule core was not well centred, reflecting a production problem (see below). The wall structure consisted of a thin outer skin, a macroporous sublayer and a thick, dense inner layer (Figure lb). However, unlike 75% HEMA capsules”, holes (most
microcapsules:

G.D.M. Wells et al.

occasionally in the outer skin. Glycerol precipitated capsules (0.01M Tris buffer) were generally similar to those made in PBS, with a slightly greater degree of macroporosity. Those made without buffer or with 0.001M Tris [Figure 2) had dense walls, with no macroporosity and small cores (i.e. thick walls). AS before” these changes in wall structure were reproducible (within this work] and were presumed to reflect the effect of precipitation conditions on the rates of polymer precipitation and gelation. The microencapsulation apparatus was designed so that one capsule would form for each dip of the coaxial needle into the hexadecane overlayer (Wmin). Unfortunately, consistent results could be obtained only with one droplet for every two cycles. Flow rates were adjusted over a wide range, the polymer solution concentration was lowered to 9% to lower viscosity, and the coaxial needle was adjusted to centre better the inner needle, to enable operation at the desired 30 droplets/ min. These adjustments were successful occasionally but not consistently. After formation capsules were tacky, with a propensity for sticking to one another and to the Petri dish. Gentle agitation or minimal time at rest were required to prevent agglomeration of some of the capsules. Stickiness decreased gradually over time and by 7 d was generally not a problem, presumably because of slow rearrangement of the polymer in the wa1114.

Urea nitrogen

a

617

assay

Figure 3 shows that the hepatocytes encapsulated with a PBS precipitant produced urea, although to a lesser extent than the control monolayers. All capsules and monolayers were incubated in RPM1 1640 with supplements. The appearance of hepatocytes cultured in monolayers was correlated with urea production rates to some extent. The deterioration in urea production paralleled the visual deterioration.

b Figure 1 Scanning electron micrographs of HEMA-MMA capsules precipitated in PBS. a, Fractured capsule; b, capsule wall.

Figure 2 Scanning electron micrograph of capsule precipitated in 0.001 M Tris buffered glycerol solution. Biomaterials 1993, Vol. 14 No. 8

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Figure 3 Urea production from 0, microencapsulated and 0, control hepatocytes based on hepatocyte concentration in input to encapsulation needle and assuming 100% encapsulation efficiency. Capsules precipitated in PBS. Mean +s.d., n = 3 batches of capsules or control hepatocytes. Control cells were the same isolates as encapsulated cells.

Figure 4 shows urea production by capsules precipitated in the isotonic glycerol precipitant which was buffered with 0.01M Tris. As with PBS, encapsulated hepatocytes produced urea but again at a lower rate than hepatocytes

cultured in monolayers. On the other hand, hepatocytes encapsulated using the isotonic glycerol precipitant buffered with 0.001 M Tris produced urea only on the first day following encapsulation, not on following days.

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Figure 4 Urea production from 0, microencapsulated and 0, control hepatocytes based on hepatocyte concentration in input to encapsulation needle and assuming 100% encapsulation efficiency. Capsules precipitated in isotonic glycerol. Mean ks.d., n = 3 batches of capsules; mean + range, n = 2 for control cells. Control cells were from same isolates as encapsulated cells. Biomaterials 1993, Vol.

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Unbuffered precipitated day 1.

of viable hepatocytes in HEMA-MMA

glycerol capsules

[initial pH 7.4, final failed to produce urea

pH 6.5) even on

DISCUSSION Encapsulation Despite the success in encapsulating hepatocytes there were areas where the microencapsulation system could be improved. Walls were thicker than desired, leading presumably to greater diffusion limitations. The polymer solution to cell flow rate was greater than that used for rate was half the other cells2’, the capsule formation design rate and the cores were frequently off-centre. Capsules cured faster here, perhaps because of the presence of collagen and sodium alginate in the core solution, This may have led concomitantly to greater attachment of the polymer solution to the needle tip through premature precipitation of the polymer at the needle, where it comes into contact with the aqueous core. The off centring appears to be related to the need for the polymer solution to seal at the ‘top’ of the capsule, as it is detached from the needle and this is very sensitive to the care to which the coextrusion needle is assembled prior to each run. Altered needle tip shapes appear to circumvent much of the latter problem in current studies. The role of capsule core viscosity on capsule formation has been documented18. PBS precipitated capsules were slightly more macroporous than before22, perhaps reflecting the more rapid precipitation of the capsule wall. This more rapid precipitation may even account for the <1 pm holes that were found frequently in the outer surface of the capsule; similar holes were not found previously. Unexpectedly, however, the Tris buffered glycerol precipitated capsules had dense walls with little macroporosity unlike the macroporous capsules prepared before using unbuffered glycerol: this probably reflects the slower precipitation conditions observed here. Presumably then these capsules did not have the increased permeability, consequent to increased macroporosity, that was observed before. Cell viability Although the amount of urea synthesized by encapsulated hepatocytes was less than the amount synthesized by control hepatocytes, it can still be concluded that the hepatocytes survived encapsulation and remained viable for at least 2 wk. However, the number of encapsulated cells was based on the cell suspension flow rate and the cell concentration in the cell feed. Physical loss of cells during capsule curing was expected to occur and this would result in an underestimation of cell number per capsule. This loss has been quantified in the encapsulation of CHO cells’e~‘7 and pancreatic islets15, but was not done here. Viability of hepatocytes during encapsulation was not measureable and day 1 urea nitrogen results were used as an estimate of day 0 viability. We note that there was no significant difference on day 1 between encapsulated and control hepatocytes in PBS and glycerol precipitated capsules. Other factors that can be used to explain reduced urea production are cell death during encapsulation and

microcapsules:

G. D. M. Wells et al.

619

reduced production rate per encapsulated cell due to microenvironmental changes. Permeability constraints are possible although a mathematical model suggests cells could have that this is unlikely l4 . Alternatively, been adversely affected by the presence of PEGZOO, by space constraints or by the inappropriate surface character of the HEMA-MMA polymer (despite the added collagen] as a substrate for hepatocyte attachment and growth. Further study is necessary to define which of these many factors are relevant. In general, encapsulated hepatocytes survived for as long as unencapsulated hepatocytes maintained in conventional culture, with the progressive deterioration in encapsulated cell viability paralleling that for the control cells. For the future, better methods for culturing hepatocytes will be necessary; then, more thorough investigations of encapsulated hepatocyte function will be warranted.

CONCLUSIONS Viable rat hepatocytes were encapsulated in a HEMAMMA copolymer and were capable of producing urea for at least 2 wk after encapsulation. Deterioration in encapsulated cells urea production paralleled that of unencapsulated cells in conventional culture on collagen indicating that culture techniques will need to be improved before this encapsulation method can be investigated as a means of restoring liver failure.

ACKNOWLEDGEMENTS We acknowledge

the financial support of the Natural Science and Engineering Research Council, the Medical Research Council and the Ontario Centre for Materials Research.

REFERENCES Chang, T.M.S., Hemoperfusion over microencapsulated adsorbent in a patient with hepatic coma, Lancet 1973,2, 1371-1372 Freeman, J.G., Matthewson, K. and Record, CO., Plasmapharesis in acute liver failure, ht.1.Artif. organs 1966,9, 433-438 Shu, CD. and Chang, T.M.S., Tyrosinase immobilized within artificial cells for detoxification in liver perfusion: II. In vivostudies in fulminant hepatic failure rats, Znt. J. Artif. Organs 1961,4, 62-64 Matsumura, K.N., Guevara, G.R., Huston, H., Hamilton, W.L., Rikimare, M., Yamasaki, G. and Matsumura, MS., Hybrid bioartificial liver in hepatic failure: preliminary clinical report, Surgery 1967,101,99-103 Demetriou, A.A., Whiting, J.F., Feldman, D., Levenson, S.M., Chowdhury, N.R., Niscioni, A.D., Kram, M. and Chowdhury, J.R., Replacement of liver function in rats by transplantation of microcarrier-attached hepatocytes, Science1966,233,1190-1192 Olumide, F., Eliashir, A., Kralios, N., Norton, L. and Eiseman, B., Hepatic support with hepatocyte suspensions in a permeable membrane dialyzer, Surgery 1977, 92, 599-606 Vacanti, J.P., Morse, M.A., Saltzman, W.M., Domb, A.J., Biomaterials

1993,Vol. 14 No. 8

620

8

9

10

11

12

13

14

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microcapsules: G.D.M. Wells et al.

of viable hepatocytes in HEMA-MMA

Perez-Atayade, A. and Langer, R.S., Selective cell transplantation using bioabsorbable artificial polymers as matrices, J. Pediatric Surg. 1988, 23, 3-9 Thompson, J.A., Haudenscbild, C.C., Anderson, K.D., DiPietro, J.M., Anderson, W.F. and Maciag, T., Heparin binding growth factor 1 induces the formation of organoid neovascular structures in vitro, Proc. Nat1 Acad. Sci. USA 1989, 86, 7928-7932 Sun, A.M., Cai, Z., Shi, Z., Ma, F., O’Shea, G.M. and Gharapetian, H. Microencapsulated hepatocytes as a bioartificial liver, Trans. Am. Sot. Artif. Intern. Organs 1988, 32, 39-41 Sun, A.M., Cai, Z., Shi, Z., Ma, F.and O’Shea, G.M., Microencapsulated hepatocytes: an in vitro and in vivo study, Biomater. Artif. Cells, Artif. Organs 1987, 15, 483-496 Dixit, V., Gordon, V.P., Pappas, S.C. and Fisher, M.M., Increased survival in galactosamine induced fulminant haptic failure in rats following intraperitoneal transplantation of isolated encapsulated hepatocytes, in Hybrid Artificial Organs (Eds C. Baquey and B. Dupuy), Vol. 177, Colloque INSERM, Paris, France, 1989, pp 257-264 Wong, H. and Chang, T.M.S., Bio-artificial liver: implanted artificial cells microencapsulated living hepatocytes increases survival of liver failure rats, Znt. 1, Artif. Organs 1986, 9, 335-336 Dixit, V., Darvasi, R., Arthur, M., Brezina, M., Lewin, K. and Gitnick, G., Restoration of liver function in Gunn rats without immunosuppression using transplanted microencapsulated hepatocytes, Hepatology 1990, 12, 1342-1349 Douglas, J.A. and Sefton, M.V., The permeability of

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Eudragit RL and HEMA-MMA microcapsules to glucose and inulin, Biotechnol. Bioeng. 1990, 36, 653-864 Sefton, M.V., Kharlip, L., Horvath, V. and Roberts, T., Controlled release using microencapsulated mammalian cells, I. Conk. Rel. 1992, 19,289-298 Uludag, H. and Sefton, M.V., A calorimetric assay for cellular activity in microcapsules, Biomaterials 1990,11, 708-712 Uludag, H. and Sefton, M.V., Metabolic activity of CHO fibroblasts in HEMA-MMA microcapsules, Biotechnol. Bioeng. 1992, 39, 672-678 Uludag, H. and Sefton, M.V., Metabolic activity and proliferation of CHO cells in hydroxyethyl methacrylatemethyl methacrylate (HEMA-MMA) microcapsules, Cell Transplantation [in press] Babensee, J., De Boni, U. and Sefton, M.V., Morphological assessment of hepatoma cells (HepG2) microencapsulated in a HEMA-MMA copolymer with and without Matrigel, J. Biomed. Mater. Res. 1992, 26, 1401-1418 Enat, R., Jefferson, D.M., Ruiz-Opazo, N., Gatmaitan, Z., Leinwand, L.A. and Reid, L.M., Hepatocyte proliferation in viva: its dependence on the use of serum free hormonally defined medium and substrata of extracellular matrix, Proc. Nat1 Acad. Sci. USA 1984, 61,1411-1415 Stevenson, W.T.K., Evangelista, R. and Sefton, M.V., Preparation and characterization of thermoplastic polymers from hydroxyalkyl methacrylates, J. Appl. Polym. Sci. 1987, 34, 65-83 Crooks, C.A., Douglas, J.A., Broughton, R.L. and Sefton, M.V., Microencapsulation of mammalian cells in a HEMA-MMA copolymer: effects on capsule morphology and permeability, J. Biomed. Mater. Res. 1990, 24, 1241-1262

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