CA2034633A1 - Alginate microcapsules, methods of making and using same - Google Patents

Abstract

Improved Alginate Microcapsules Methods of Making and Using Same Abstract A composition for containing biological material for in vivo implantation and transplantation comprising alginate cross-linked with barium salt, preferably barium chloride. The microcapsule may optionally have as its other layers hyaluronic acid and poly-L-lysine. Also alginate cross-linked with both calcium chloride and barium chloride may be used. The microcapsule of the present invention is rugged and retains a strong negative charge, enhancing the release of protein and limiting the invasion of immunoglobulins. The microcapsule may preferably be used for encapsulating islets of Langerhans for the production of insulin.

Description

2~3~633 hQ~E e~ M ~ a~ ~iDsL~
~hi~ i8 ~ continu~tion of co-pending applic~tion U S
Serial No 07~445,983, filed D~cember 13, 1989 ~his invention relates to the fields of polymer chemistry, lmmunolsgy ~nd transplantation, and more particul~rly to tho field of materials ~or use in conjunction with transplant~tion and implant~tion of foreign cell~ and biological materials 2 A~ Ba~ Qun~
Evid~nc~ xi~t~ that transplantation of in~ulin-producing c~llc (i-l~t~) can cur~ the di~b~tic ni~l of the need for insulin therapy Th- ma~or ob~taclo pr-v~nting cl$nical ~ucc~es in isl-t tr~n~plantat$on a- a therapy for diabete~ to date h~ b-en immunogoniclty of tho cQll and r-~-ction oS tho tran~pl~nt-d gr~ft 8urvival of i~let ~llografts and or x-nografte h~s b~en ~chieved by v~rious ~thod~ of $mmuno~uppr-seion and/or r-lated immunological t-chniqu-e ~owev-r, euch t-chniqu-~ h~vo bad only llmit-d ~ucc--- in th~t th- tr~n~pl~nt-d ielet c~116 curviv- only a ~hort while b-fore r-~-ction occure ln ~dd$t$on, th- xt-ndod u-e of i~munoeuppr--~iv- g-nte Dften r ~ult~ in ~-v-r- oomplic~tione, uch ~ r~n~l d~m~g~ and v-n c~ncer in th- tr~n~pl~nt r-cipiont On- colution to thi~ probl-m of groft r-~-ction i~
the introd~ction Or ~ phycic~ omi-p~rm-~bl- b~rrier b-twa-n th- tr~n~pl~nt-d l-l-t- ~nd the ho~t'- immune ~y-t-~ by th- ~-thod o~ microoncop~ul~tion Microonc~p~ulation i~ ~ proc-~ in which mall, di-cr-te ~, . . ~. . ................... .
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~at~rialc, vi~bl- b~ologic~l ti-~ue or c~llc, liguid dropl-tc, or ga~-s ~ro compl~t-ly nv-lop~d by an lnt~ct membrAne which 1- pr-f-r~bly compatible w$th the b~ological y~t-m in ~hich it i~ plac-d ~he ~unction of the ~icroc~psul- membrane i~ to prst~ct th~ m~terial within from lmmunological r-co~nition by th- host and to control the flow of ~t-ri~l~ in-ide ~nd out~ide the microc~psul- ~cro~s the m-mbr~ne A lArg- body of llt-r~tur- on ~icroenc~p~ul~tion hac b~en produc-d lncluding D~rquy, S ~nd Reach, G
~ia~etol~ia, (1985) 528 776-780; Lim, F ~nd Sun, A
Sc~çnQ~, (1980) 210 908-910; Lim, F ~nd Mocc, R
of l~ 9seut~caL_~ie~ç~l (April, 1981) 351 - 354;
O'Shea, t ~1 Bioch~mic~ et Biç~hysica ~ctal 804 (1984) 133- 136; Leung, et ~1 ~ (1983) 7(2) 208-21 2; Ar~ki, ot ~ , Vol 34, Sopt-mber 1985, 850-854; ~nd U S P~t-nt Noc 4,690,682; 4,4~9,331;
4,391,909, ~mong other~
In ~ddition to iclet cellc, othor m~terialJ ~uch ~c microbial c-llc, other mamm~ n cell-, yeastc, chloxopl~stc, pl~nt protoplacts, mitochondriA ~nd nzymos h~ve been l~mobilizod ~nd ntr~pped ucing ~icro-nc~psul~tion techniques Among th~ ~teri~l~ u~ed in nc~psul~tion ~r- calcium ~lgin~t- 9-1~ Llm ~nd 8un, ln 1980, cucc~ ully micro-nc~pcul~t-d i-l-t~ u~ing algin~t- g-l, poly-Lrly~in- and poly-thyl-nimine The nc~p~ulat-d i-l-t~ w-r- ln~-cted lntr~p-riton-~lly into di b-tic r-t- Th- anim~l~' blood gluco~ v-l- dropp~d to norm~l for two to thr-- ve-k~, cugge~ting th~t th- nc~p~ulation proco~ h~d prot-ct-d the $~1-t- from lnva~$on by th- r-cipicnt~' l~mun- ~yct-m However, th~e ~tudio~ how-d th~t th- ~icrocapsul-- wore v-ntu~lly r-~-ct-d as a r-~ult o~ ~ibro-i~, or t~brobl~t form~tlon ~round th- ~icrocap~ul-, which v-ntu~lly ro~trict~ the flow of nutri-nt~ to th~ c-ll~ cont~ined $n ; the ~icrocap~ul- and th- outflow of ~teri~l from the ~icroc~p~ul-~ cr-~t-d by th~ t cell~ di~po~ed therein - ,, "~', ~ ' ' ' . ~ .
-2~4~33 Tho Lim ~nd Sun c~p~ul~c ar- ucually ~ade by firct formin~ a n-gAt~v-ly chargod alginato b-~d around purifi-d ~nd i~ol~t-d icl~t c~llc by ero-~-linking algin~t- with caleium ehlorido, then eroating a positiv~ly eharged ~mbran~ on the outer ~urfacQ by forming an ionic bond with a c~tlon ~uch ac poly-L-lycin- ~ddit~onally, ~eond negativoly eharged outer lay~r of ~lqinat~ ic usu~lly formed around the outcide of the poly-L-lycine layor, lonie~lly bonded ther~to Finally, th- inner b~ad of algin~te i~ d-gellQd, le~ving a eapcul- currounded by ~ layer of poly-L-lysine-algin~te gel and an outer layer of alq~n~te Thic prior ~rt capcule ie depictod $n Figure 1 and d~scribed in more detail below Cap~ules formed according to the foregoing procQdure are diffieult to make, requiring many ctepc, whieh is not adYantagQouc in light of the eoncidQration that live eells are involved Alco, it ic d~cirablQ to minimiz- handling time and moder~to handling eonditionc Even ~ore ~ignificant, how~vQr, ic thQ faet that the~- prior art eapcul-c oSt-n tail in vivo as a r--ult Or th- r-lea~e of ~ub~tane-fi whieh ~timulate eytokin- r-l-ac-, whieh in turn eau~- th- ~ieroeapsul-- to b- ~ttaek-d by immunoglobulinc The immunoglobulin~ ~y ither, or in eombination, penetrat- the mieroeapculo and de~troy tha neloc-d islet e~ , eauce fibroblact formation around th- mieroeapcul-thereby ehoking off nutri-nt~ to th- eollc and pr-v-ntlng the e-ll produets from b ing r-l-a--d ~nto th- boct;
~nd/or ~timulat- th- d-ctruetion of th- ~lerocapcul- vi~
th- ho-t'- lmmunologieal ~y-t-m Alginate, th- prineipal mat-r$al of th~
~ieroeap~ul-e, ic a b-t-rog-n-ouc group of lln-ar bin~ry copoly~-rs o~ link d p-D-m~nnuronlc ecid (M) and itc C-5 pimer ~-Lrguluronic acid (G) ~h- ~onom~r- ar-arrang-d ln a blockwi~- patt-rn along th- poly~-ric chain wh~r- homopolym-ric r~gion- are int~r~pac-d with ~-qu-nc~s containing both ~onomQr~ ~h- proportion and c-qucntial arrangem~nt of th~ uronic acids in alginat- d~pend upon ,, . - .. ,- - :
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the cpocie6 of algae and the kind of algal ti~uo from which the ~at-rial i~ pr-par-d V~riou- properti~s of diff-r~nt typec of ~lginat~ ar- ba--d upon th~ guluronic acid Dakeup oS th~ particular alginato For xamplo, viscocity depends mainly upon th- mol-cular ~iz-, wh~r-as th~ ~ffinity for dival-nt ions ~-s~ntial for the gel-forming prop~rti~ are r-lat-d to th~ guluronic acid content 8p-c$fically, two con~-cutiv- di-~xially linked G r~sidue6 provide binding ~it-- for c~lcium ions ~nd long -quences of ~uch ite~ form croc~-links with ~imilar ~equences in other Alginate mol-cule6, giving rice to gel network6 .
~ t has bo~n d~monstrat-d that a cign$ficant ~timulant to the roloaso of cytokines i6 the 1-4 linked ~-D-mannuronic acid (M) componont of alginate ~See copending pat-nt application Sorial No 468,905 ~h-se M
blockc do not bind with calcium when the gel i8 formed in tho inner b-ad, and it is boll-v-d that omo of this M
alginate l~ache~ out aftor tho microcapsul- i~ for~ed ~n accordance with the th-orios in th- prior ~rt, it ha~ traditionally been belicv-d t~at microcapsules form an ~ffectivo barri-r again~t immunoglobulin p-n-tration by having a rufficiQntly cmall diam-ter porosity that large protein6 ~r- xcluded How-ver, it m~y bo that the nogative charge of the alqinate bead plays a more cignificant rol- in xcluding n-gatively charged protoins, ~uch ~ immunoglobulins ~ h~ preoent invention ov-rco~o the defici-ncico ot the prior art by pro~iding ~ microcapcul- of th- typ-da~cribed herein ln accordanc- wlth the following d~cription, ~ well ~ ~ncillary mat-rialc ~nd r-thodo r~l~ting th-r-to ~ummarv of th~ Invention Th- pr-~-nt inv-ntion provid~ ucce-o~ul ~ppro~ch to microencap~ula~ion and lmplantation which hac not heretofor- b--n di-cov-red : .

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2~3~33 It i~ on~ ob~-ct of th~ pr-sent inv-ntion to provid~
a ~t-ri~l which m~y b- impl~nt-d or tr~n~pl~nt-d ~ y~yQ
which i- non-i~munogenic ~nd non-~lbrobl~ct lnduc~ng It i~ y-t ~nother ob~-ct o~ tho pr~--nt inv~ntion to provlde a ~icro-nc~p~ulation ~y~t-m utilizing alginat~
which i~ gell-d ucinq barium salt ln~t-~d of th- prior ~rt c~lcium chloride It i~ ~nother ob~-ct of the inY~ntion to provid~ a ~icro~nc~p-ul~tion ~ystem ln whlch the ~lgin~te b~d r~mains in a g-ll-d ~t~te It i~ y-t another ob~-ct of the pr~sent inv~ntion to provide ~ m$crocapfiul- which ic mor~ r~gg~d ~nd dur~ble than prior art c~p~ul-s, and which r~tains ~ gr~ter n~gatlve ch~rge over a longer period of time than prior art microc~pcul-c It i~ ~nother object of the pre~ent invention to provide a microoncap6ul~tion system which decreace6 immunogenicity r-l~tiv~ to prior ~rt c~psul-~ by limiting tho l-aching of M block ~lginate 8till anothor ob~ect of the proc~nt inv-ntion ic to provide ~ ~icro-ncapsul~tion ~ystem wbich incre~ces immunoprotoct~bility of th- cont-nt~ th-r-of by lncr-~6ing ~nd ~aint~ining the nsgativo ch~rge in the cor-, thereby prev~nting or ~lnimizing the ntry lnto s~id microc~p6ule of n~g~tlvoly ch~rg-d lmmunoglobulins j Yet ~noth-r ob~-et of th- pr-s-nt inv-ntion 1- to i provid- a mleroencap-ulation y~t-m ~ith i~prov-d in~ulin or oth-r prot-in or produet r-l-~-- eh~r~et-ri~tie-r--ultlng ~ro~ th- n-g~tiv- eh~rg- in th- c~p~ul-Th- pr---nt lnv-ntion eo~prl~-~ a n-w neap~ul-tion ~aterial eompri~-d of lgin~t- g-ll-d by b~rium ~lt, th-~torial being uceful ~n YiYQ for lmpl~nt~tion ~nd tr~n-pl~nt~tion ln ~mm~ n bodl-~ Th- ~t-r~ ay t~ke ~any form~, ~uch a- ~h-et~, org~n eap~ul~tion and the lik~, but 1~ pr-~r~bly u--d ~or ~iero ne~p~ul~tion of li~ing e~ wbieh ~r- for-ign to th- ho~t ln whieh they ar~ l~pl~nted ~hR pr-~-nt inv-ntion al-o prot-et~ the .
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~ 2~3~633 ~clets of Langerhans or other tran~pl~nted ti~u~ from i~munoloqical cell xejection The pre~nt inv-ntion ~lso provldoc a microQncap~ul~tion y-t~m which lim~tc fibrobla~t ov-rgrowth In on~ mbodiment, the presQnt inv-ntion relat-6 to ncAp~ul~t$on of cells or othQr biologic~l ~at-riAl ~ith a coating of algin~te gell~d ~ith b~rium ~lt, prefQrably, barium chloride Optionally, ~ c-cond lAyor of poly-L~ly~ine, and a third out-rmost layor of elqin~te, may be add-d to the capsule The alg$n~t~ in th- outer coating $c pr-fer~bly comprised o~ subctantially quluronic acid, with minor amounts of mannuronic acid block6 In ~ ~econd embodiment, the algin~te portion of either of the former ~mbodiments ic gelled with combination of barium ~nd calc$um In yet another ombodiment, the inner layer of the microcapsule is comprised of barium g-ll-d or barium plus calcium gelled ~lginate, which i~ then coated ~ith poly-L-lycine and ~n outer lay~r of hyaluronic wid Other ~mbodiments, and the d-tailc of th- pr-sent invention will be best underctood with refer~nc- to the drawing~ provid~d herewith and descr$bed bri-fly b-low Brief Descri~tion of the Drawings FICURE 1 1- nn illustr~tion of a cro~c-~-ction of the prior ~rt ~icrocapcul- d-picting the v~riouc lay-r~ ~nd one ex~mpl- of th- potential cont-nt- of th- ~$crocap~ul~
FICURE 2 i~ ~n lllustration of a cross-~ect$on of one mbod$ment of th- pre~ent inv-ntion FICURE 3 is nn illustration of ~ cros~ ction o~
~notb-r mbodim-nt of th- present inv-ntion FIGURE 4 i~ ~n illu~tr~tion of ~ cro~ qction of the preforred ~bodim~nt of th- pr-~ent invention rICURE 5 ic ~n illuctration of a cross-soction of ~noth~r mbodi~ent of th~ pr-c-nt invention . . .

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2~3~33 The pr-~ent invention co~pri~e~ ~t~rial ~hich can be lmplant-d or tran~plant-d in yivo lnto na2mal- without induclng ny ub~tantial i~munog-nic r-~ction or fibrobl~t tormation Th~ pre~ont inv~ntion ~l~o eompri~e~ rial~ for ncap~ulation o~ biological ~atorial~ Th- pr-~-nt lnv-ntlon ~- al~o a proco~ for miero~ncapculating biologic~l e~ and oth-r ~t-ri~l~
tor u-- in implant~tion or tran~pl~ntation ~ ~ drug or biologieal mat~ri~l deliv-ry ~y~t-m AB u~d h-r~in, the term biologic~l mat-r$al- include~ prokaryotic and uk~ryotic cell~ wh$ch ~r~ ith~r n~tur~lly occurring or genetically ~ngino~red, druq~ or pharm~coutic~le, nzymes, part~ of cell- uch ae mitochondria and protoplaete or any other naturally occurring or synthesized m~t-rial which may be implanted The ~at~rial used in the pre~ent in~-ntion i~
alginate cros~-linkod with barium ~alt, ~nd pr-f-r~bly barium chloride ~he ~lgin~t~ may bo ny lginato ~olution c~pable of forming microc~p~ules, as i~ known $n the art Ihe alginate m~y b~ eompri~ed ub~tantially of ~-L-guluronic acid (G) whieh may b~ ref~rred to h-re$n a~
quluronic acid alginate or high C
The u~e of high guluronic acid alginate is de~cribed in our eop-nding p~t-nt application 8erial No ~46,~62 8mall amount~ of mannuronle eid (~-D-~annuron$e cid) are al~o pr-~-nt Th-r- ~r- ~t l-~-t 65% C r-~idu-~ or mor~, and pref-rably about 85% C r--idu-~ ~nd 15% or 1-~ M
r-~idu-~ ln ~iqh C ~lginat- Alglnat- eo eompri~d licit~ ~ v-ry low r--pon~- trom ~onocyt-~ in the production or tumor n-cro~i~ factor ~TNF) and IL-l and ILr 6, ~nd, ~8 a r-~ult, do-- not aliclt fibro~1- 8uch ~lginat- ~ay b- obtain~d rro~ Prot~n A/S, Dramm-n, Norway High C ~lginat- i- th- pr-f-rr-d ~lginat- u~-d on th~
Dut~lde of ~icrocap~ul~- b~cau-- of it~ prop rty of not inducing f~brobla~t for~tlon , ;. ' .
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2~134~33 Figur~ 1 hows the prior art c~p~ul~ o~ Li~ and Sun As ~hown in ~igur~ uch ~rior art microcap~ul~
compri~e l~l-t~ o~ Lang-rhanc 12 or oth~r subst~nce for tran~plantation or $~pl~ntation contain-d ln ~ l~quid bead S Gr c~pRul~ of ~lginate 14 which was g~ d with c~lcium chloride during the Dak$ng of tb- m$crocap6ul- ~nd then ungell~d to r-turn it to a liguid t~te Surrounding the calc$um-~lginat- liqu$d be~d $~ y-r o~ ~oly-L-lysine 16 which for~s a ~embrane by bonding lonically w~th the ~lginate core On the outside $s ~nothRr l~yer of ~lginat~ 20 A~ ~hown in Figur~ 2, the pr-~ent inv~ntion, $n one mbodi~ent, comprises islets of Langerhans 12 or other transplantation or implantation ~aterial, coat-d with ~
bead of alginate 22 gelled with a barium ~alt, pr-ferably barium chloride Ac hown ln F~gure 3 the i~l-t of Langerh~n~ ~2 may be ~urrounded by ~ b~rium ~lginat~ gQl co~ting 22, e8 in Figur~ 2, which in turn i~ surrounded by a poly-Lrlysine lay~r 16, which $n turn ~8 ~urrounded by an outer layer of ~lginate 24, pr-ferably high G alginate A~ ~hown $n Figure 4, which depicts the px-~-rred Qmbodi~ent, the $~1et of L~ngerhans 12 ~ay be ~urrounded by ~n ~lginat~ gel coating 26, that $s gellod w$th both calcium ~nd barlum, which $n turn $- urround-d by ~
poly-Lrly~ino layer 16, which ln turn $- urrounded by an out-r layer of alginate 2~, pr-f-rably high C alginate A~ ~hown $n Figur- 5 th- $~1-t of Lang-rhan~ 12 ~ay b- curround-d by a b~rium alginat- b-ad 22 or an alg~nat-b ad gell-d with both calcium and bar$um 2C, w~ich $n turn i~ ~urroundod ~y ~ poly-Lrly~ine lay-r 16, which $n turn 1- urroundod by ~n out-r l~y-r of hyaluronic ~cid 30 Thu~, th~ barium ~lginat~ cap~ule may be u~ed alone or in con~unction with other lay-r~ to form ~icrocapcule In the pref-rr-d mbodim~nt, a 1 0% to l S%
by w~lsht alginat- ~olution $~ ~oroed ~round puri~iod ~ t~ of Langorhan~ ~nd 1~ tr-ated wlth a olution in the .. . . .
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203~633 r~nge of 2 to 20 ~M bariun chloride to ~orm a gelled ~icrocap~ul-~ n ~noth-r ~bodiment, ~n ~lgin~t- b-~d havlng a conc~ntration o~ ~ 0 to 1 5~ alginat- 1~ Pir~t tr~at~d S with a ~olution o~ 80 to ~00 ~M calcium chlorid-, to bind th~ G-bloc~, and then with a ~cond olution Or 1 to 20 ~M barium chloride to bind the blocks o~ th- alglnate compo-ition In y~t ~noth-r mbodi~nt, th- ~icrocap~ul- ot th~
$mm~diately ror-going ~mbodiment 1~ ~urtber tr-ated with a ~olution o~ 0 5% poly-L-ly6ine (20,000 MW) An outer coating of 1 1% alginate, pr-f~r~bly h$gh G ~lg~n~te, i~
thsn form~d therearound Ac ~ middle lay~r, poly-L-ly~ine i~ the preferred material However, lt will be appreciated by a p~r~on of ordinary ~kill in the ~rt that poly-L-ornithine ~nd chitosan may be u~ed in place of poly-L-ly~ine, and that oth~r catlonic compounds with similar properti-6 ~ay ~l~o b- u~-d ~h- u-~ of hy~luronic acid a~ a compon-nt of th-pr--ent inv-ntion inhibit~ th- ~orm~tion o~ ~broblast~
when appli-d a- an out~ido coating on th- microc~p~ul-Th-r- ar- ~any improv-m~nt~ provided a~ a r-~ult of the pr-~ent inv~ntion Fir~t, barium alginat- tend~ to be a mor- ruqg-d ~nd hardy mat-rial than prior art calcium alginat~ Al~o, f-w r ~t-p~ ar- r-quir-d in the ~anuf~ctur- ot barium alginat- Dicrocap~ul~, fir~t becau~- nultipl- lay-ra r not r-quir-d, and al-o, 1~ ~
~any lay-r~ ar- u--d, th-r i- no n--d ~or a d--g-lling t-p ~- 1- ua-d ln th- prior art Wh-n alginat- bead- ar- tr-at-d with both barium chlorlde and calcium chlorid-, th- b-~d i- rir~t dropp-d ln a olution of calclu~ chlorid~, and then in a ~olution o~ bariu~ chlorid- ~h- calcium i- b-li-v-d to cro-~-link with th- guluronic acid block- of h- alginat- Jol-cul-s, and th~ barium cro-~-link~ both ~ith th- M-block portion~

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of th~ ~lginAt~ ~nd tbe C-block ~ortionc which hzv~ not previouYly been cro~s-link-d with th- calcium chloride The r-Eulting ~icrocap~ul-s of th- pr~nt lnv~ntion hav~ ~mprov-d kinstic~ of in~ulin r-l-~e ~h~ b~rium chlorld~ ~ol ~t-rial h~ ~ gr-at~r n-gativ- charge b-cau~- it i- in a ~-1 form, r~th-r tban a l~quld form, and alco b-cau~- ov-r tim~, the liguid c~lcium ~lgin~t- of th~ prior Art microc~p~ul-- le~ch-- out o th-re i~ c n-gatively charg~d m~teri~l in prior Art microcapcul-s The n~qativ~ly charged portion r~pelc the negatively charqed in~ulin, or other n~g~tiv-ly charged material theroby forcing ~id inculin or oth-r m~tori~l out of the microcap-ul- Conv-r~ly, th- n-gativ- charge would ~l~o repel immunoglobulin molecules produced by the host, thereby ~afely protecting the cont~nts of the microcapsulo For $n vivo ~pplication~ of the pre~ent inv~ntion, the compo~ition compri~ing alqinate having ~ high G
content ~ay be u~ed in the form of organ capculation, heetc of ~lginate for impl~nt~tion, hollow fib~r~ and membranes formed of the ub~-ct composition Exam~le 1 Sinale-layer Microenc~sul~tion of Isle~s of Lan9~b~n~
Cultur~d rat i-l-tc of L~ng-rhan~ (2 X 103 l-l-tc in 0 2 ~1 m~dium) may b~ ~u~p nded unifor~ly ln 2 ml of a l S% (w/w) odium ~lginat- olution (~i~co~ity 51 cp~) ln phy~iological ~line 8ph-rical dropl-t- cont~ining ~ t~ ~-r- producod by ~yring- ~ump~air ~-t xtru~ion through a 22-gaug~ n-edl- ~nd coll-ct-d in 1 5% (wJw) b~rium chlorid- olution Ib- ~up-rnat~nt w~ d-c~nt-d ~nd th- g-ll-d ph-rical ~lgin~t- dropl-t~, containing ~ t~, w-r- w~h~d with dilute CHES (2-cyclohcxyl~mino-than- cul~onic ecid) ~olution ~nd 1 1~ barium chloride olution Th- ~icrocap~ul-~ ~r- found to b- gen~rally ~pheric~l ~nd ach to cont~in ~rom 1 to 2 vi~ble i~l-t~ The ~ , .:

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2~3~633 microcap~ul-~ have a diam~t-r oS 500 t 50 ~ ~nd wall thickn~ of ~bout 3-4 ~m ~h- ~lcroc~psul~ y be ~uspQnd~d in nutri~nt ~dium ~t 37 C

S Mut~ layer Microen~ ul~Qa o~ l~le~5 of LanQerhan~
Cultured r~t 1-lst~ of Lang-rh~n~ (2 X 103 i-l-tc in O 2 ~1 ~odium) ~ay be ~u~pond-d uniformly in 2 ~1 of ~
1 5% (w/w) ~odium alginat~ ~olution (vi~co~ity Sl cp~) in phy~iological ~aline Sphoric~l dropl-ts containing i~l-tc wor~ producad by ~yringe pump/~ir ~et ~xtru~ion through a 22-gauqe n-edle and collectod in 1 5% (w/w) ~rium chloride ~olution The ~upern~tant was d-cantod and the gell-d ~pherical alginate dropletc, cont~ining i-l~t6, were wash~d with dilu~e CHES olution and 1 1% b~riu~
chloride ~olution Aft-r ~pirating off th~ ~upernatant, tho gelled dropl-t~ w-re incubated for 6 ~inut-s in 0 05% (w/w) polyly~ine h~ving 4 ~olecul~r w-ight of 17,000 The aupernatant Wa8 d-canted ~nd th~ polyly~ine c~p~ul-- w-r- wa~hod with dilut- CHES, 1 1% calcium chlorid- oolution ~nd phy~iological ~alin~ Th~ wa~h-d polyly~ino cap~ul-~ w~re incubatod for 4 ~inuto~ in 30 ml of 0 03% odium alqinat~ to p rmit ho rOrmation of ~n out-r ~lginat- ~-mbr~n- on tho lnitial polyly~in-~-~bran-, by lonic int-r~ction b tw-on th- n-q~tiv-ly charg-d algln~t- ~nd th- po~itlv-ly charg-d polyly~in~
The alginat- u-Qd in th- out-r co~ting, and i~ d-~ir-d, th~ inn-r co~ting ~ w-ll, 1~ poly C algin~t~ (Protan) produ¢-d a- d--crib d ~bov-Th- ~icrocap-ul-- ar- ~ound to b p-r~-ctly ~ph-ric~l ~nd ~ch to cont~in ~rom 1 to 2 vi~bl- l-l-t~ Th~
~icrocap-ul-~ b~v- a dia~-t-r Or 700 ~ 50 ~m nd w~ll tbickn~ Or about 5 ~m ~h- nicroc~p~ul-- ~y b-u-p-nd-d ln nutri-nt D-dium ~t 3~ C

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Exa~L~

Cultured rat 1~1-tc Or Lang-rhan~ (2 X 103 lcl-t~ ln 0 2 ~1 ~-dlum) w-r- ~u~p-nd-d unlfor~ly in 2 ml of a 1 5%
(w~w) ~odium algin~t- ~olution ~vi-co~ity 51 Cp6) in phy-iologic~l ~al$n- Sph-ric~l dropl-t- contain$nq ~ ts w-r- produc-d by ~yring- pump/air ~ot xtru-ion through a 22-gaug- n--dl- and coll-ct~d in 1 5% (w/w) calcium chlorid~ ~olution ~he ~up~rnatant wa~ docanted and the gell-d ~ph-rical alginatR dropl-t~, containing i61ets, were then coll~ct~d in 1 5% ~w/w) barium chloride ~he ~upern~tant was ~gain d~canted and th~ gelled ~pherical alginate dropletc w-re washed with dilute CHES
lS Golution and 1 1% calcium chloride ~olution Aft-r aspirating off th- ~up~rnatant, the gellod droplots were incubat~d for 6 minut~s in 0 05% (w/w) polyly~ine having ~ molecular weight of 17,000 ~ he ~up~rnatant wa6 decant~d and th~ polyly6ine capsul-s were wa~hed with dilut- CHES, 1 1% calcium chloride ~olutlon and phyc~ological ~lin~ ~hQ wash~d polylysine cap-ul-~ were incubat-d for 4 minutes ln 30 ml of 0 03% ~odium alglnate to permit th~ formation of an outer ~lginat- membrane on the initlal polylycine ~-mbrane, by lonic lnt-raction botw-on th- n-gatively chargod alginatQ and th- po-itiv-ly charg-d polyly-in~
Th- ~lg$nat- u~-d in th- out-r coating, ~nd lr d-~lr-d, the inner coatlng ~- w~ll, 1- poly C ~lginate produced dQscrlb-d abov-Th- microcap~ul-- ar- found to b- p~rf-ctly ~ph-rlcal ~nd ach to contain ~rom 1 to 2 viabl- icl-ta Th~
~crocapcul-~ would ~av- ~ dia~-t-r of 700 ~ S0 ~m and wall thickn~ o~ about 5 ~m Th- microcAp6ul-~ may be ~uspQndQd $n nutri~nt m-dium at 37 C
It w$11 b- obviou~ to ~ p-r~on of ordin~ry ~kill in th~ art that th~ pre6ent inv-ntion i~ not limited in ltc ~pplicDtion to ~p~cific b$ologic~1 material6 to be ~ . . . .
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, . , . -, : -, ., 2~3~33 ~ 3 ~ncapsulat-d, ruch a~ the i~l~t c~ d~ rib-d in d~tail ~bove, or ~y th- ~p-cifically d~crib-d oth~r inn~r l~y-r-o~ microcap~ul~ cu~d h-r-in ~h- only limit~tion~ of th~ pr-~nt lnv-ntion are ~et rort~ in the cla~m~ appQnded h~r-to ~nd ~ny guival~nts ther-o~

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Claims (16)

1. A transplantation or implantation material comprising biological material encapsulated with a coating material comprising alginate cross-linked with barium salt.

2. The material of Claim 1 wherein said biological material comprises purified islets capable of producing insulin.

3. The material of Claim 1 wherein said biological material is microencapsulated in a first innermost layer of barium chloride cross-linked alginate, a second intermediate layer of cationic material selected from poly-L-lysine, poly-L-ornithine and chitosan and a third outermost layer selected from alginate comprised substantially of .alpha.-L-guluronic acid with minor amounts .beta.-D-mannuronic acid and hyaluronic acid.

4. The material of Claim 3 wherein, said second layer is poly-L-lysine and said third layer is alginate comprised substantially of gelled .alpha.-L-guluronic acid with minor amounts .beta.-D-mannuronic acid.

5. The composition of Claim 4 wherein said poly-L-lysine comprises a molecular weight of less than 20,000 daltons.

6. The composition of Claim 1 wherein said alginate is cross-linked with barium chloride with calcium chloride.

7. A method of encapsulating biological material comprising forming around said biological material a bead of alginate gel cross-linked with barium salt.

8. The method of Claim 7 wherein said biological material comprises islet cells.

9. The method of Claim 8 wherein said islet cells are first microencapsulated in a bead of barium cross-linked alginate gel, then encapsulated in a layer composed of material selected from poly-L-lysine, poly-L-ornithine and chitosan, and then encapsulated in an outermost layer comprising material selected from hyoluronic acid and alginate comprised substantially of .alpha.-L-guluronic acid with minor amounts .beta.-D-mannuronic acid.

10. The method of Claim 9 wherein said second layer material is poly-L-lysine and said outermost layer material is alginate comprised substantially .alpha.-L-guluronic acid with minor amounts .beta.-D-mannuronic acid.

11. The method of Claim 10 wherein said poly-L-lysine comprises a molecular weight of less than 20,000 daltons.

12. The method of Claim 7 further comprising cross-linking said first layer of alginate with both barium salt and calcium salt.

13. A method of making a microcapsule for containing islets of Langerhans comprising forming around said islets of Langerhans a bead comprised of alginate, and gelling said alginate with barium chloride.

14. The method of Claim 13 wherein said alginate is in a concentration in the range of 1% to 1.5%.

15. The method of Claim 13 wherein said barium chloride in the range of 2 to 20 mM.

16. The method of Claim 13 further comprising treating said microcapsule with calcium chloride in the range of 80 to 100 mM.