MICROENCAPSULATION OF LIVING CELLS IN COVALENTLY CROSSLINKED ALGINATE-POLY-L-LYSINE-ALGINATE MEMBRANES

摘要

Immunoisolation of cells in semipermeable membranes has been proposed as ameans to prevent their immune destruction following transplantation[1, 2].However,several fundamental issues remain to be addressed before considering clinicalapplicationof this method. One such issue is the resistance of microcapsules to chemicalandmechanical degradation. Strong microcapsules will obviously increase thedurability ofthe transplant. It is also likely to improve long-term biocompatibility ofmicrocapsules,since a strong pericapsular reaction always develops around broken or damagedcapsules[3, 4]. Moreover, the strength and durability of microcapsule membranearecritical issues for the transplantation of virus-transfected bioengineeredcells. Thesevirus-transfected bioengineered cells potentially include insulin-producingcells as well as cellsproducing molecules, which promote islet cellsurvival/replication/transdifferentiation orinsure islet and microcapsule immunoprotection. Finally, considering thelimited supplyof human islets, the most promising approach to .beta.-cell replacement is thetransdifferentiation of stem cells into islets[5] (not necessarily embryonicstem cells,which arise controversy, but stem cells of many other origins, includingautologous cells).Note that even autologous cells would require immunoprotection against therecurrenceof the autoimmune process. The major concern related to this approach is therisk ofmalignant transformation of immature stem cells. Microcapsules that can hardlybe destroyed in conditions compatible with life would provide a safe method oftransplanting stem cell derived cells, bioengineered cells or immortalizedcells.Complexation between alginate and polycations such as poly-L-lysine(PLL) isthemost widely used method to microencapsulate cells[2]. Microcapsules areconstructedby a simple three steps procedure. First, the calls are entrapped in a beadformed bythe ionic cross-linking of alginate by a divalent ration such as calcium[6].The secondstep comprises coating the alginate beads with a polycation such as PLL, whichformsa peripheral membrane ensuring a good control of the molecular weight cut-offandincreasing membrane stability. Finally, the third step consists to coat themicrocapsules with a dilute alginate solution for insuring biocompatibility.The twolatter reactions rely on the ionic interactions between the polyanionicalginate and thepolycationic polymer of lysine[7, 8].In order to improve microcapsule strength, we[9] and others[7, 8, 10, 11],haveevaluated the effect on microcapsule strength of modulating intrinsicparameters PLLmolecular weight, concentration and incubation time and the mannuronicacid/guluronicacid ratio of alginate. The formation of neutral capsules by the introductionof a newcoating agent[12] has also been investigated. Following these experiments,tighterbinding between PLL and alginate was obtained. Nevertheless, poly-L-lysinestillcompetes with other positively charged molecules in the environment to bind tothealginate beads. A prolonged incubation in solutions with high concentrationsof Ca2+ orSr2+ has showed a displacement of the alginate-poly-L-lysine bounds[13, 14].In addition,Thu et al. have observed the progressive loss of the external sheet ofalginate frommicrocapsules within days or weeks[7].To prevent this competition with charged molecules in the environment, a newconcept has been proposed: the introduction of covalent links into themembrane of themicrocapsule[15-17]. Covalent links are known to be more stable thanelectrostaticinteractions. The challenge is that most methods to create or to break acovalent link areincompatible with the survival of living cells. The objective of the presentwork was todevelop and validate a method, compatible with encapsulated cell survival, tocreate acovalent link between the PLL layer and both alginate from the microcapsulecore andfrom the outer sheet. It would be unlikely that such a microcapsules would bedestroyedin conditions found in the living body.Here we show that it is possible to microencapsulate living cells in membranescomposed of covalently linked alginate-PLL-alginate membranes, withoutaffecting cellviability. The formation of a covalent link between PLL and alginate from boththealginate core and the outer alginate shoes considerably increased microcapsuleresistanceto chemical and mechanical stress. Standard APA microcapsules dissolved within45seconds when they were incubated in an alkaline solution whereas covalentlylinked APAmicrocapsules remained unaltered after two years in the same alkaline solutionCovalently linked microcapsules were 22 times more resistant than standardmicrocapsules when they were submitted to a mechanical stress. The process isbased onthe chemical derivation of poly-L-lysine (PLL) with a photoactivatableheterobifunctional cross-linker, N-5-Azido-2-nitrobenzoyloxysuccinimide (ANB-NOS),which is able to covalently bind PLL to alginate when energized by UVA lightillumination[18, 19]. The N-hydroxysuccinimid of the cross-linked was fistcovalentlylinked to PLL in conditions that would damage living cells, but before thecolts areinvolved in the procedure. Then islet cells encapsulated in calcium alginatemicrocapsules were incubated with the arose-linker derived PLL, than inalginate again toform the outer biocompatible sheet. When the preparation is illuminated with aUVAlamp the photoactivatable residue reacts with the phenyl azide residue onalginatecreating a covalent link between PLL and alginate. The latter reaction is notharmful forliving cells. All reactions involving the crosslinker-derived PLL wereperformed in a darkroom, until UVA illumination, to prevent activation of the photoactivatableresidue.We, herein, present a proof that a covalent link was formed providingconsiderableimprovement of microcapsule resistance to chemical and mechanical degradation.Theresults of the study showed that the procedure did not modify microcapsulemembranepermeability and did not affect in vitro and in viva encapsulated cellsurvival.