CA2179341A1 - Methods for isolating, enriching and increasing the maturation of cells - Google Patents

Abstract

The present invention provides a novel culture system for the isolation, enrichment and maintenance of neonatal islet cells in vitro, for use in transplantation in vivo.
Cellular compositions produced by the culture method are also provided, including mocroencapsulated cells useful for transplantation, for example in diabetes. Also provided is a method of increasing the maturation rate or proliferation rate of a cell utilizing microencapsulated techniques. The invention also provides a method of treatment of a subject having diabetes utilizing cells produced by the culture method described herein.

Description

U~17~ IU.'{ 1;~_25 F~}. ~19 ~78 509a FISH & RIC~4RDS01',' P.C. ~005 A~, orr.~y ~c~!~et:.'J725~3~

METHO~S FOR ISOL~TlNG, ~NRIC~rING A~D I~CREASING TE~
MAI'UR~TION OF CELLS

~I~LD O~ TH~ ~TI~i~
~rhe prese~t inver~tion xel~tes g~e~ally to methods .or culiuring and m~turating cells, in particular, neGnata islet cells. The3e celLs are usefl:l Lc~r t~znsp' ~tation, . o~
exarnple, into a subjec' suffering ~ror.~ a ~iabetic con~itio~.
These transp7 anted cell~ supp~ emen' or rep' ~ce the di~betic sub je~t ' s de~icient pancreatic islet ~ells, all~wing the patient to p~oduce insulin in ~e~ponse to glu~,oce with~u~
inslli~ ir.jections.
BACKGROUND OF TH~ TTON
Irlsu~ ir.-dependent diabetes mellitus ~Type I) is 15 . callsed by the progressi~Je destruction of -he insulin-produc-in~ pancreatic islet ~eils, which e~entu~lly leads to l fe-long dependence by t:~e diabetic s~lbject or patient on insul in 'cherapy. A major focus of diabetes researc~ has been to ~evelop a better treatment to correc t ~r alleviate the symptoms ~f dia~:ietes, thexe~y p~eventir.g t~e disabl~rlg complications or the disease. One cpproac;~ is o ~ransplanl isolated insulin-producin~ pancreatic islet cells i~tc~ the dia}: etic sub jec~. Cl- nical ~ri21s usiY~g certa_r types of slet transplantation ha~e corrected _he a~r.ormall y-high D' OO~ glucose levels in Type ~ dia~etic~, ren~e~ing the tr~nsplart recipients partially or to~ally ir.dep ~de~t c exogenous ins~lin ther~y. For the pa~ients whic~. zre now indapen~ent cl tre ins~lin therapy, the tr~nsp1cr.tcti.Gn has rest~ed e~glycemia. Se~eral ~ases ha~e been rep~rted lA

OB~17i~ YON 13:20 FA~ 78 50~ FISH & RICH~R~SON P.C. I~IOO~

herein human isiet allotranspl~t~tion has corre~ed baSa1 hyperglycemia, ~endering the recipients i~sulin-in~epen~en~
for va~yirl~ perio~ of time ~1-6). This ~ece~lt succes~ is attributable to the developme~t of rep~oducible ~etho~s for lsolatin~ and p~rifying ~uman islets (7-g) and the p~o~ucti~n of an ade~uate ce~l mass to achieve ~nsulin-indepen~e~ce. If isle~ transp'antation is to be~ome ~ widesprea~ treatmert ~cr Type I diabetics, howe~er, the supp~y of ~onor organs ~ust be increase~.
~o o~er~o~e this supply pro~lem, islet tissLe fro~
abunda~t and a~cessible anim~l sour~es i~ keing co~si~ered ~or xenotransplantation (10-17~. Pigs meet the necessary requirements 'or a xe~oger.ei~ source of insulin-p~oducing tis~ue as they br~ed r~pidly, h~ve l~rge litters, ~n~ exhibit ~orpholo~ical and physiolo~ical chara texistics C~LParab1e to humar.s. ~or~ine ins~ is ~tru~turally similar to human insuli~ an~ has been safely used for treating Type I hum~n ~iabe~ics vi~ ex~genous insulin treatment.
Unfortunately, des~ite ma~y reports o~ the isol~-tion o~ ~dult porc~ne isle~s, fa~tors, ~uch a~ age, breed, and qual~ty o~ organs, adversely affect the final yield ~18, lg). A~ul.t porcine isle~s are frag~le, ~ifficul~ to isolate, difficult to m~int~in in tissue culture ~17, ~C, 21) and difficult t~ ~ryopreserve. ~he ~ragility o~ ~hese adul~
islets also signi~icantly dee~eases the yields o~ islet cells p~rticularly when culture pro~edures are used to ~e~uce gr~ft i,mmunogenicity or when low ~emperature s~orage i~ used to combi~e isol~tes from ~ulti~le ~onors. I~ contr~st, tissue culture o~ a G~liagenase-digested felal po~ine p~r~c~eas produ~s viable islet-like cell clusters tlo~l2~ 14), which have the ability to cure ~ eti~ n~de ~i~e within two months postt~ansp;an~tion ~11, 12~. Howe~er, generally, r~t ~22-0~17~ MON 13: 2B FA~ ~19 ~78 50~ FISH ~ RICHARDSON P.C. 1j~007 21 7q341 .

25), porcine (ll, 12~, and human l26) ~et~l p~ncre~ic çe1ls exh~bit ~ poor i~s~lin secretory response tc glucose (~2-Z~), a~d the onset an~ ma~uration of glucose-induced insulin sec~etion is more e~i~en~ in the postna~al period (~-25).

a S~Y O~ T~E I~ENTION
The present inven~ior is bas~ upon the discovery OL a ~ovel cult~re sys~em fo~ the e~richment of selecte~
celis in vitro. In one em~odi~ent, the invent~on pr~vides a~
in vitro method of isolating ~ su~s~antially enr1ched ;O population of cells fro~ a tissue source, ~he me~no~ compris-ing;
~) treating the tissue sour~e to form ~ p~epara-tion of cellsi ~nd b) cult~ring the preparation of a~ in ~.tro ~ith ~ ser~m-free basal medla suppleme~ted with ~ serum sub.~ti-~llte, ~n agent to stimul~te ~NA synthesis, and ~n agen~ to promote endocrine cel~ sur~ival, r~sulting in ~ s~bstzntially enriched popul~t~o~ of cell~ from the tissue source. Mo~t preferred is the ~se of a porcine pan~re~s as the tissue source an~ e~docrine cell~, e.g~, insulln-secreting ~eonat~' islet cells, a$ the subst~ntially en~iched popul~tion of cells.
In another embodimen~, the in~ti~n pro~ides for a novel culture ~edium, which comprise~ a serum-~r2e basal 2S ~edi~ suppleme~te~ with a serum substi~ute, ~n ~en~ to stimulate ~NA ~ynthesis, and ~n agent to promote endocrine cell su~ival. Prefer~ly, the serum substitute is ~lbumin, the DNA-synthesis stimulating agent is i~obutylmethylxanthene ~IBMX) and~or nicotinamide, and the end~çrine ce~1 sur~iv~l agent is gl~cose and/or IBMX.

0~17~ 0~ 13:27 FA~ el~ ~78 509~ FISH ~ RICHARDSON P.C. 1~lO08 ln a further embodiment, the invention pro~ides an enriched popula~ion of endocrine cells, pxeferably porcine rLeonatal insulin-s~cre~ islet cells, pro~u~ed by the in ~i~ro culture metho~ described ~ove. This population of S cells a~so c~n be ~ranspl~nted into a suitable recipient, su~h as a human ~iabetic patie~t, and optionally can be transfected with ~n exogenous gene prior to transpl~ntation.
In a ~till further e~bodiment, the in~en~ion pro~ides a ~ethod for in~reasing the rate of proli~eratio~ of a cell co~prising en~psul~ting the cell within ~ stabilizing matrix an~ cultur~g the resulting cell in vitro. Also, the ~ate of matura~ion o~ an undifferenti~t~à cell can be inGreased ~ he encapsulation of that cell. Preferably, ~he cell can be encaps~lated withi~ ~ microcapsule to for~ a microe~c~psula~ed cell. Typically, the microc~p~ule com-prises algin~te o~ a~arose, which forms a thin l~yer o~
material ar~und the encap~ulated cell. I~ one ~spect of ~he invention, ~ore tha~ one ~ell type is encapsulated within the microcapsule.

~0 Brief DescriDtion of the ~rawinae Figure 1 shows an electron ~icrograph of por~ine NrC
aggregates after nine da~s culture. Beta cells appear well granula~ed, s~ructu~ally intact and contain sec~e~ory ~5 gra~ule~ whi~h confo~ to ~dull gra~ule morpholo~y. The aggregate also ~ontains no~-granul~ted ~ells ~ nd num.erous d~ct-like stxu~ures ~arrows~ (x130~.

Figure ~ shows li~ht micro~raphs of rLati~e neon~t~ 3 day ol~ por~ine pancreas ~A,~ ~250), ~ day ~llltured ~I~
~ggregates ~C,D x~50), a~d porcine NIC ~ggregates ;4 weeks Oa/17/lla l~lON 13:27 FAl~ e78 soa~ FISH & RICIIARDSON P.C. I~boo~

~ .

after transplant~tion be~ea~h the kidney sap~uLe ~ alloxan dia~eti~ nude ~i~e ~E,F x125; g,h x25~). Se~tions were ~our.ter~tained with H~r~is's hemotoxylin then ~ oh~soch~i~ally stai~ed fo~ insulin (A,C,E,G~ or gluc~gon 'B,D,F,~).

Figure 3 shows bloo~ glucose values duxi~g oral (~5 OGTT) a~d intraperit~neal ~B: IPGTT) ad~inistra~ion of glucose to nu~e mice tr~nsplanted wilh 1000 ~- ; n-10~ or ~OOO ~- ; n=1~ por~i~e NIC aggreg~es in comparison to age-~atched normal control mice (~; ~=9). *~0.05, tPCC.01 vs. norm~l controls, $Pc0.05 V5 . 1000 a~gregate re~ipients Fi~ure 4 sho~s elect~on microsccpy of porcine NIC
aggregates 14 ~eeks af~er transpl~ntation ber,ea~h the ki~ney capsule ~f alloxan dia~etic nude ~ice. ~ cells are stru~tur-ally i~t~ct, highly granulated ~nd contain well developed endop~asmic reticulum; some cel~s a~e shown to 6xpress mitotic figures (arrows, x 6~00j.

0~17~ llON 1~: 27 PA2t ~ 78 50~ FISH & RICHARDSON P. C. l~l O10 ~SCR~PTIO~ OF TH~ P~EFERRED ~B~IN~.~T
The invention pro~ides ~n in vitro ~e~hod ~or enriching a populatlon ~f pre~erred cells from a ti~sue source co~prisi~ tre~ting ~he tissue source to form a preparation a~ ~hen cul~ring the prepar~tion in vi~ro with a serum-free basal ~edia supple~ented with a ser~ ~ubsti-tute, an agent to stimul~te ~ synthesls, a~d an ~gen~ to promote survival of the selected cell type, thereby xesulting in an en~i~hed population of ~esired cells. The selected cell type can inelude, ~t is not limited to, ~iffere~tiated en~ocri~e cells, such as neuroendocrine cells an~ a~xenal cell~, panc~e~tic endrocrine cells, endocrine .precursor cells, such as ~tem cells ~nd du~t cellsi hepatocy~es, ~d the llke. Preferably, the cells enriched are ~ifferentiated 1~ e~doc~i~e ce~ls or endocrine precursor cells, with i~su~in~
secreting neonat21 lsle~ cells ~ost preferred.
The source of tis~ue can ~e ~ ype so long as it ~o~tains the selected or ~esire~ cells for enrichment, ~n~/~r corLtains precursor cells cap~ble of beco~i~ the desired 23 cells d~ring the cul~ure process. For exa~ple, ti~sue canL
include all or part.of the iiver, pancre~s, thyroid ~land, xeproducti~e gl~nds, myoc~rdial ~issue, ren~l ti~sue, 3100d, ~n~ the like, or a presele~te~ gro~p of cells from within these or~an~. Mos~ preferred as a tissue source is ~he ~5 p~n~reas, whereby it is isol~ted fro~ ~ pig or h~man.
Further, the sour~e o~ the pa~creatic ~issue can be pre-adolesce~t, fetal or neo~atal, with ~eonat~l as ~he most preferre~ source.
The enriche~ population o~ prefer~ed cell~ is substantiall~ enriched ~s described herein~ The term "s~stan-tially enriched" as used herein means ~ population of selected cells wherein the majoLity of or ~t le~st ~bout ~0 o~ the cells are the selected ~ell type. For example, enri~hed aggregates of ~eonatal islet ~ells Gontain a~o~t 5 0~17~ MO~I 13:28 FA~ 878 50~ FIS~ & RICE~RDSON P.C~ lnll - 2 1 7q34 1 or less fi~roblasts or pancreatic exocrine ~ells and most p~eferably contain about 2-54 or le~s of s~h cells.
rn the present l~Lethod, the tiss~e is ~pically prepared }:~y partial or full di~estion, a$ necessary, using an agent or mixture of agents, su~h a~ protease~, ~oll a~en~e~, other enzyI[es, su~h as disphase, ~ he like to form a ~ di~e~. Colla~en~se, su~h as Type ~ a preferxe~ a~ent and car~ be obtained comrnerGially f~om various ven~ors, s~ch ~s Sigma, Ser~7a and E30ehringer ~annheim. Othe~ ~ollagen~ses C in~lude li~erase, Type ~, and T~pe XI, which also ca~ be pur~hase~ commercially. Alter~a~i~e~y, de~endi~g on the ti sue source ~lsed, it may be desirable to use physic~l methods o~ preparing the enriched popul~tion ~f cells. For ex~ple, when blood i~ ~he tissue source, i~ ~ay ~e desi~ble to use af fin l ty chro~natography .
3uring the ~ulture ~te~ of the process, a sexum-free basal media supplemen~ed with various .aG~ors or ~gents is utilized. The agents inclu~e, but ~re n~t li~ited to, a se~u~ substitute, an agent to sti~late DNA sy~thesis, ~nd ~n agent to prom~te cell s~rviv~l ~f the d~si~ed cell type. The media must ~e serum free; serum i$ omitte~ from the media due to i~s a~ility to pr~mote the survi~1 of conta~in~ti~ or undesl~e~ cells, su~h as fibrobl~sts, p~ncre~tic exocrine ce~ls, and the like. Therefore, the seru~ substitute must not promote, ~n~n~e/ or pro~iferate ~ conta~i~ati~g cell type. ~ypically ~nd most preferred, ~l~umin is u~ d as the serum sub~titu~e, with k~vine sexum ~lbumin bei~g ~he most common.
~n ~ge~t or agents ~p~ble o~ st_mui~ti~g ~NA
synthesis irl the sele~te~ celL type or precur~or is a~e~ to the media as ~ s~pplement. For ex~mple, isobutyl-methyl~an~hene (IBMX) ~n be used for its capa~ity to stim~late DNA sy~thesis i~ islet cells and its polenti~l to e~hance the survi~l of cultured islets. Another ~N~-synthesis sti mulating ~gent is nicotinamide, wherein it h~s 0~/17~ 0~ 13:28 FA~ B78 509~ FISH ~ RICHAR~SON P.C. 0012 21 7~34 1 the abiiity to sti~late ~slet cell DNA ropl.1~ation an~ to positively affect the metabolic function of fetal or ~eonatal islet cells. ~lev~ted glucose co~entration~, i.e., greater than or equal to l~mmol/liter, a~so stimul~te DNA sy~thesis.
Also i~cluded is a supplement in the ~edi~ is an agent capable of promoting ~ell survi~l o~ the cell ~esired fo~ enrich~ent or c~pable of pr~tecting the de~ire~ cell from destruction durin~ the c~lture process. For e~am~le, gluoose and/or IBM~ can be used as the ~ell survival pro~ctin~ agent iO ~ue to its cytoprotective effe~t of islet cells ~uri~
Gulture and its a~llity to enh~nce i~let cell replication.
The preferred supplements are prese~l a~ about Lhe followin~ ~oncentrations ~n the me~ia; ~l~u~i~ from abou~
O.1~ to aboul 1 . 0~ weight/volu~e; INBX from about 5 to about 100 ~mol~liter; ~icotina~ide fro~ abou~ ~.5 rO abo~t ~0 m-mol/liter, and glucose fro~ about 6 to abcut 30 mmol/liter.
The di~est is typicall~ cultured at a temperature from ~bout 20~ to a~out 3g~C for a~out 7 to about ~0 da~s in h~i~ified ~ir. Most prefe~red, the digest is cultu~ed ro~ abou~ 9 d~ys at a temperature of abo~t 37~C.
The ser~m-free tissue culture o~ b~l media is commonly used liquid tiss~e cultuxe media that is free of sexum. The media cf the invention ~tilizes the~e me~ia in comoination with ~elected supplements or ~o~ponents to ç~q~te a no~el ~edia to cul~ure, enri~h and allow the proliferation of, ~or example, the islet cells in ~itro. ~asal me~ia useful in the ~uiture method o~ the inventi~n i~ ~ny serum-free tissue culture media know~ in the art, in~ ing Media 19~ ~Gibco), CMR~ 10~6 ~ c~) media, H~'s F10 ~Gibco) tiss~e ~ulture ~,edia, ~n~ the like. These media also cohta n ~rious ingredients, f~r example, ~mino ac~ds, ~itamins, inorgani~ salts, ~uf~ering ~gents, and ener~y sou~ces.
Purified molecules, such ~ hormones, gr~wth factors, transport proteins, trace elements, anti~ioti~$, ~nd O~J17~ 10~ 13:28 F~ 78 50~ FISH & RICEIAR~SON P.C. 1~lol3 -substratu~-modifying ingredients optionally ~an be in~luded in the me~
~n a prefexred e~odimen~, the inve~tion comprises ~ se~um-free bas~l me~ia supple~e~ted with albumin, IBMX, ~icotina~i~e, ~n~ glucose. The pan~reatic digest from or~e or more neonatal pig pan~re~s is preferably cultured with H~ml~
F10 tiss~e culture media, suppleme~ted with abou~ 10 m moltliter o~ glueose, about 50 ~mol~liter o~ IEMX, abou~ 0~5~
weight~olu~e of bovine seru~ al~umin (~SA), and a~o~t 10 mmol~litex of nicotinamide. The ~e~ia can opt~Gn~lly ~e supplemented with amino a~idsr such as ~-glu~a~ine, and ~n~i~ioticst such as.penicilli~ ~n~ streptomycin. The amolnt ef ~-qlu~a~ine is a~out 2 mmol/liter, whereas the ~mount v~
penicilli~ and of s~reptomyci~ is ~out 100 T~/ml a~d 100 ~gJml respectlvely.
Once the su~sta~tially e~riched popul~tion of cells is produced by the culture, it can ~e u~i~ize~ as descr~e~
kelow ~r the çells ca~ be further isolated, purified/ or mature~. .
~he present in~ention pro~ides methods fo~ ~ultur-ing a sour~e of isle~ cel~s for use in transplan~ n. On aver~ge 250,000 islets are recovered from an 80-gram adult human pançreas, where~s, the instant inventio~ allows the isol~tion of abo~t 50,000 islets from ~ ~-3 gr~m neo~atal pig 2~ p~ncreas~ The res~ltir.g neon~tal porcine islets ~n be t~ansplanted in~ diabe~i~ $u~ects; thereby impro~in~ their long-term prognosis.
In a preferred embodiment, a large-s~le method of isolati~g neonatal islet Gell ~NIG) aggregates from 2 neona~al pancreas ls pro~i~ed. The metho~ com~prises digest-ing the p~ncreas with collagenase; a~d cultl~rin~ the digest with a serum-free tissue cul~ure or b~l medi~ s~pplemented with glucose, IBMX, albumin, ~ nicotinamide at a~out 3~~C
i~ humidified air for ~out nine ~ys: ~esulting in ~eona~a~
islet cell aggregate~. The sour¢e of the p~ncre~s can ~e .9.

0~,'17~ MON 1~: 2~ FA~ 878 50~ ~ISH &~ RICHARDSOr~ P. C . E~l 014 ~ 7934 1 from any a~imal, including a hum~, but pox~ine p~ncrea~es are p~eferre~.
The islet ~ells as described herei~ ~re useful for ~ene thexapy methods as well, An exoge~ous or foreig~ gene o~
choi~e can be tranferred to the is~et cells prior ~o tr~ns-plantation. An 'rexo~e~o~s" or ~'foreig~" gene refers to gene~ic material fro~ outside the islet ~ell which is introduced into the cell. The term also in~lu~es a gene that has ~een modifie~ from the native or n~tural form of a gene found i~ ~he cell to be tr~nsfected. For example, variouS
cyt~kine genes or ~he like ~a~ be ~rarsfecte~ int~ the ~ells ~y methods o~ tra~s~eGtion as calcium phosph~te co-precipi.
~ation, conve~tion~l mechanical proce~ures su~h ~5 ~icro-injection, biolistics, insertion of a plasmid encased in 1-iposo~e~, or by use of viral vec~ors. For ex~mpie, one method is to use a eukaryotic viral vector, s~lch as simian virus 40 (S~4G) or bovine papil~om~ virus, to transiently in~ect or transfo~m the neuroblast (~u~a~yotic V~ral ~ectors, Cold Spring H~r~or L~boratory, Gluz~an ed~ 82~.
Varlous vi~al vectors which c~n be utilized or transfer of genes to the isle~ ¢ells a~ taught herel~ include ~denovi-rus, herpes virus, vac~i~ia, and preferably, an RNA virus such as ~ retro~irus. Retroviruses are ~seful p~rt cularly in the case of ~ivici~g cells. Therefo~e, the ~etho~ of the ~5 in~ention, w~ich provi~es a ~eans for produGing divi~ing and~or differentia~ed neonatal isl~t celLs, provides ~ells that axe sus~eptible to ret~o~ ~ i~fecti~n. Prefer~ly, the retro~iral vector is A derivative of a ~urine or avian retro~irus~ E~amples o~ retroviral ~e~tors 1~ wh~ch a single fo~e.ign gene c~n ~e inserted incl~de, ~ut a~e no~ limited ~o:
Mol~ney murine leuke~,ia virus ~MoMu~3, Har~ey ~.urine sarcoma uirus ~HaM~SV), muri~e ~m~ry tUmor vir~s (~ TV)~ and Rous Sarcoma Virus ~RSVl. For human ~e~ls, preferably gibbon ~pe leuke~,ia virus (~LV) is utilized. A number of ~dditional r-etroviral ve~tors ~an ihcorporate multiple genes. All of -lo-OB~17t~ ON 13: 2~ FA2~ ~la ~78 50~ FISH & RICEIAR~SO~ P.C. 1~1015 2 ~ 7934 t these ~ectors can transfer or incorporate ~ gene .o~ a s-elec~a~le mar~er so th~t transduced ~ells ~an be identified a~d genera~e~.
- Since recombinant retroviruses are de fe~tlve, t~ey ~equire assis~ance in order to pro~uce infec~ious ve~t~
parti~les. Thi~ ~s~istançe ~an be provided, for example, by using helper ~ell lines that contain pl~s~.ids encodi~ all o~
the s~ructural genes of the retrovlr~s (gag, e~v, and pol genes) under the control of regulatory sequences wi~hin the long termin~- xepeat ~LT~. These plasmi~s are missing a nucieotide seq~ence which enable~ the packagi~ me~h~ni~ to reGognize ~n RNA ~xans~ript ~or encapsidation. Helper cell lines which have deletion~ o~ the pa~kaging signal inclllde, but are not li~ited to ~2, PA317, PA12, CRIP and CRE, for exa~p~e. These ~ell lines produ~e empty ~irions, since no ge~ome is pack~ged. If ~ retroviral vector is introduced into ~uch cells in whLch the pack~ging signal is i~tact, ~ut the struc~ur~l genes are repl~ced by other genes of interest, ~he ~ector can b~ packaged and ~ector v~rion produced. The 23 vector ~irions produced b~ thi~ method ~an th~n ~e use~ lo in~ect a tissue cell ~ine, such as ~IH 3T3 cells, to p~oduGe 1arge ~uantit~es of chimeric retrovir~l virions~
Alternati~ely, NIH 3T3 or othe~ ~issue culture cells can be dixectly t~ansfected ~ith pl~smi~s encoding the retro~iral ~5 structural genes gag~ pol and e~v, ~y ~onventional ~çium phosph~e or lipofection transfection. These cells ~re then tr~ns~ected with the ~e~tor plasmid cont~inin~ the ~enes of interest. The resultin~ cells ~e~ease the retrovir~l ~ec~or into the cultu~e ~edium.
The in~ention also envisio~ production of tra~sgeni~
ani~als, e.g., pigs, which would produce a~ unli~i~ed ~pply of islet ~ells ~or transplantation. Cells removed from su~h pigs are cultured and isolated prior to transplantation ~ ~allotra~splan~s) by the metho~s desçribe~ herein. ~See US
Patent No. 4,7~,B~6; EP 247,4g4~

-Il-O~J17~ ION 13:30 FA~ ~la ~78 50~ FIS~ RIC~ SON P.C. Ii~lol~

The ir,vention also ~escribes a method of treating a subject h~ing ~iabetes by ad~i~istering to the ~u~ject a therapeutically-effe~tive ~mOUnt ~ islet cells, such as transplanting the in ~i~ro i~sulin secretin~ neonat~l islets descri~ed herein. "~her~peutiçally-effecti~e'l as ~sed herein, re~ers to th~t ~ount of islet cells that is of s~uffiGient quantity to alleviate a symptom of the dise~e or to ~melio~ate the dia~etic disorder. "A~iel~orate" refers to ~essening or lowerlng the ~isease's ~ ~isor~rls detriment~l effect in the patient receivin~ the t~erapy. In the ~ase o~
~ di2~etics, the treatme~t can lower o~ eliminate the depend-enc~ o~ exo~enous insulin.
The diabetic subjects or pat~ents to 'oe tre~ted include ~;m~l 5~ su~h as sheep, pigs, cats, xodents, ~attle, non-human primates, and most pre~er~bly dog~ an~ hum~ns. The culture methods of the inventi~n provide a source o~ neo~at~l islet ¢ells from hum~ns or othex spe~ies, such as bo~ine, sheep, pigs, or non-h~man primates, wit~ pi~s and humans as pre~erre~ s~urces.
The invent~on alsc des~ribes ~ethods ~or tr~nsplan-~ation, typic~lly of xenografts, but also of allog~afts.
Such methods include culturing ~eon~tal islet oells to e~xi~h islet endocrine cells (differentiated and~or undifferenti ated), and then transpl~nting the resuLti~ ~ells into a ~uit~ble recipient. Glucose toler~nce t~sting, ~ ~e~ribe~
hexein, can be used to monitor the effe~tivenes~ of the islet tr~nspl~nt.
Any of the transplan~ation or impl~ntatlon pr~ce-dures known in the ar~ can be utilized. Fox e~ample, the selected cell~ or cells of interest can he ~urgically i~pl~nted ln~o the recipient or su~ject. Fux~her, the cells ca~ be admini~te~ed ln ~n enc~psulated for~ or ron-e~c~p~u-lated form, Transplantation or impla~t~ion is ~ypl~lly ~y simple inje~tion throu~h a hypo~ermic needle having a bore -l2-Oe~17~ lON 13:30 FA~ Bl~ e78 50~ FISH & RIC~R~SON P.C. b~l017 diameter sufficien~ to permi~ p~ss~ge cf a suspensio~ of cells therethrough without damagi~g the cells or tissue roating. For implantation, the typically encapsulated or ~ coated cells are formulate~ a~ ph~rmaceutical ~ompositions S together with a pha~mace~ ally-acceptable caxrier. Su~h compositions contain ~ sufficient n~'oer of co~ted transplant cells which can be inje~ted int~, or admi~istered ~hr~ugh ~
lapBrosçope to, an animal, usually into the perit~neal cavity if islet cells are uti~ized. Ho~ever, o~her tr~nspl~ntatio~
sites Czn be selected depen~ing up~n the cells ~nd desired biolo~ical effe~i the~e sites include the li~er, spleen, kidney caps~l~, omental pouch, and the like.
Typically, the num~e~ o~ tra~spla~ed islets is from about 5 to about 10 thousa~d per kilogra~ of body we~ght. For exa~ple~ in miCe approxi~ately 1,000 3,000 islets are transpl~nted. The number of cther cells, tissues, and the like will be cal~ul~te~ ~epen~in~ on their fun¢~ion.
While not re~uired, i~ may ~e desirable ~o adminis-~er an immunosuppressi~ ~gen~ to a recipien~ of t~e islet cells~ prior to, simultaneous wilh, ~ndJor ~ter transplant~-tion. ~ agent such as Cyclosporine A ~CsA~ i p~efera~le, however othe~ im~une suppressive age~ts can be used, such as rapamycin, desoxyspe~gualine, and like. These agents ~re administered t4 c~use an immunosuppres~i~e effect in the ~ubject, such that t~e transplante~ lslet ce ls ~re not re~ected by ~h~t subject's i~mune system. Typically, the i~m~lnnSuppreSSiVe a~e~t is ~ini~tered continuously t~ro~gh-out the tra~spl~nt treatment typicall~ o~e~ ~ period of days ~r weeks; fcr example, Cs~ trea~ent range~ from aboul ~ to about ~0 days at ~ dos~ge range of a~out 5 ~o 40 ~g per kilogra~ of body weight per da~. The agent can ~e ad~inis-tered by a v~rief~ of means, including parenteral, sub~utane-ous, i~trap~'~o~ry, oral, intr~nasal admi~i~tration and -he ~ like. Preferabl~, d~sing is gi~en by oral a~inistraticn.

0~17~ llON lJ: 31 FAl~ 78 50~8 FISH 8~ RICHARl)SON P. C. Iib 018 .. 1 , Alternati~el~, the enri~hed Ge~ls also c~n be en~apsul~e~ prior to transplantation. Although the cells are typically micxoencaps-~lated, they ~n be en~ased in ~ario~s types of hollow fibers or in a ~lock of encapsulating ! 5 ~erial. Enc~psulation provides an effective prote~tive barrier to isola~e ~he transplanted ce~s or ti~sues ~ro~ the host or recipient's immune system. TypicAl ph~rm~eu~ical encapsul~ticn com~ositions in~lu~e, e.g., liposomes, gel~tin, polyvinyl alcohol, et~yl~ellulose, cellulose acetatephthzla~e lC and s~yrene maleie a~hy~ride. Se~ Remin~ton~s Phar~aceutical S~len~e~, Mack Publishing Co., Eas~on PA ~1~90).
A varie~y of ~icroeneap~ulation methods and ~ompo~itions ~re known in the art. A ~umber of mi~roe~capsu-lation methods for use in ~r~nspl~nt ther~py h~ve focused on the use of alginate polymers or a~arose to suppl~ the en~apsula~ion ~ompoSitions. Algi~tes ~re linear polymers ~f mannuronic and guluronic aci~ residues whlsh are ~rr~ngeci ln blocks of sevex~l adjacent gu~uroni~ acid r sidues forming gulurona~e blocks and block of adj aceIlt Inannurcrlic acid 2 0 residues ~o~ming mannuronate ~loc:ks, i n~ersperse~ wi~h mixed, or heterogenous bloc~s of alternating ~uluronic and man-~uxo~ic a~id resid~es, ~ene~ally, ~onovalent c~ti~n ~lgin~te salts are s~luble, e.~., Na-al~inate.
~i~alent c~tions, such as ~a~', Bat~ or Sr~i, tend to interact with gulur4nate, and the coopera~ive 4inding of these ~t~ons within ~he guluronate blocks previ~es the pr1m~ry lntramolecular crosslinking responsible for formation of stable ion-pai~ed alginaEe ~els. Alginat~ enc~psulation metho~s generally t~ke adv~ntage o~ th~ gelling of algina~e in the prese~e of these di~lent ca~ion s~lutions. ~n particular, these methods involve the su~pension of the m~terial to be encapsulat~d, i~ a ~olution o~ monov~lent oation ~lginate salt, e.g., so~i~m. Drople~s of the solutio~
are ~hen genera~ed in air :and collected in a ~o'ution of 3~ divalent cation~, e.~., CaC~2. The ~ lent cations in~era~t 14~

"

0~17J~ Mor~ 1J:31 F~ 01~ ~7~ 50~ FlSH ~ RICHAR~SON P.C. l~bo19 -wlth the alginate at the ph~se txansition ~etween ~he droplet an~ the divaler.t cation so~u~ion resultlng in the formatio~
of a stable alglnate gel matrix ~ein~ formed. Genera~ion ~f alginate drcplets has previ~usly Deen carried out ~y a number of met~ods. ~or exa~ple, droplets have been gener~ted by extrusion of al~i~ate thro~gh a tube by gr~vltatior~ low, in~o a s~lution of div~lent ~ations. Similarly, ele~tro-~tatic droplet generators which rely or~ the ge~eration of an ele~trost~tic differential ~etween the ~lgin~te sol~ion an~
~0 the divalent catio~ solu~ion h~ve ~een described. The electrosta~ic dlfferenti~l results i~ the ~lgi~a~e solution being drawn thr~ugh a tu~e, into the s~ ion o~ div~lent ~ations. For a general di~eussion o~ dxoplet generation in encapsu~ation processes, see, e.g., M.F.A. Go~n, Fun~amen-tals of A~im~l ~ell Encapsula~ion and Immo~ilization, Ch.
pp. 1~4-142 ~CRC Press, 1~93~.
Furthe~, metho~s have bee~ descri~ed wherein droplets are ge~exa~ed from a stream of the alginate solu~ion using ~ lamin~r air flow extrusio~ device. ~pe~ificall~, thls de~ice ~omprises a ~apilla~y tube withi~ an outer sleeve. Air is dri~en through the outer sleeve and ~he pol~er solution is flow-regula~ed thro~gh the inner tu~e.
The alr flow from ~he outer sleeve ~reaks up the ~luid flowing from the capillary tube in~o small ~roplets. See U.S. Patent No. 5,2~6,495.
Microe~capsulati~n alsQ h~s been applied in the treatme~t o~ diseases ~y tran~plant therapy. While tradi-tional ~edical tre~tments ~or fu~ctional deficien~ies o~
secretory ~nd Gther bio7~gic~1 org~ns have focuse~ on repl~cing i~entified normal produ~ts of thq deficien~ organ with natural or synthetic pharmaceutical agents, transplant therapy focuses on replacement of th~t ~unction w ~h ~ell or or~an tr~nsplants. For example, the treatmen~ of insulin-dependen~ di~betes meLlitus, where the pa~creatic isle~s o~
~5 Lan~erhans are nonfun~ionai, ~an be carrie~ out by replacing 0~17/~ )lON 13: 31 FAl~ 78 50~ FISH & RICHARDSON P. /~ l 020 2 ~ 7934 1 -the normal secretion of insulin ~y the islets in the pan-creas. I~sulin m~y ~e supp~ied either b~ d~ily a~minist~a-tion of synthetic o~ su~stitute animal ll~sulin, or ~y tra~splantation of ~uncti~nal hu~an or ani~al islets.
~ttempts to transplant or~an tissues into gene~i-cally dissimilar hosts withou~ immunosuppression ~re gener-ally defeated by the immune system of the host. Accor~ingly, attempts have been made to provide effective protec~ive barrier ~oatings, e.g., by mi~roenc~p~u~ati~n, to isolate t~e ~ 10 transplant tissues from the host immune system. Successfui ~ell or tissue transplan~s gener~lly ~equ~ ~e a Goati~g th~t will prevent their de~t~uction by a hostls immune syste~, pre~ent fi~ro~i~, and wil~ ~e per~eable to a~d allow a free diffusion of the nutrients to the coated transplant and re~o~al ~f the sec~e~ory ~nd waste produ~ts from ~e coate~
transplant.
Vi~le tiss~e and ~ells ha~e been successfully im~ob~lized i~ alginate capsules coated with polyl.ysine. See J. Pha~m. Sci . 70: 351-354 ~1~81 ) . The ~se of these ~oated capsules in pancreatic islet transplantation to c~rrect the diabetic state o~ diabetic ~nir~l S h~s ~lso bee~ des~ri~ed in Science 210:~08-9Og ~1981~.
The prolcnged reversal of ~he di~eti~ state of ~i~e wi~h xenografts of microe~apsulated ~a~ islets, using alginate-polylysine capsules has ~een reported. See Dia~e~es 40:1511-1516 (~3j. The development of transplants encapsu-l~e~ in cal~ium algin~te capsules rea~ed with polylysine is also describedr for e~ample, in U~S. Patent Nos. 4~73~56~r 4,6~,293, 4,78~,550, 4,~0~,355, an~ 4J78~550~
U.S. Patent 4,744,933 descxibes encapsulating ~olu~ions contai~ing biolo~ically ~ctive m~terials in membra~e of i~er ~ea~te~ inate and polyamlno a~id.
U.S. Pate~t 4,6~6,28~ reports a method for co~ting transplants suita~le for transpl~ntation into gene~içally dissimilar in~ividuals. The method in~olves coating the Oe~17~ MO~T 13:32 FAX ~la ~78 50~ FISH ~ RICHARD50N P.C. 6~1021 t~a~spla~t with a su~fa~e ~onfoxmi~ bonding bridge of a function~l m~teri~l that ~inds che~ically to ~ surface component of the transplant, which is enveloped in a se~iper-meable, ~iologically compatible l~yer of a pol~.er th2t bin~s chemi~ally to t~e bo~ding brid~e layer.
~etho~ for introducing a sec~n~ ~lg~nate sel ~oating to ~ells ~lre~dy coated with pol~lys_r.e alginate is describe~ in U.S. Patent 5,227,~g~. Both the first and seco~d coatin~ of this method r~quire st~bili7ation by polylysine.
~ncapsul~tion methods applied ~o ~ke ~hese ma~erials have comp~ised a p~o~edure ~o~ ~orming droplets of the encapsulating ~edium a~d the ~iological ~teri~ d a pro~edure ~or soli~ifying the erl~apsulat~ ng mediuurl. Ag~rose encapsulated m~terials have 'oeen formed by chilling an emulsion of ~aaro~e droplets ~on~ining biologi~ ~a~e~ials as shown by ~ilsso~r et al., ~ature 30~ 630 ~19B3) an~
Nilsson, et al., Eur. J. App7. Mi~ro~io~. Bio~echnol. 17;319-32~ 83). Injection of ~roplets of polymer ~ont~ining bioLogi~al materials into a body of coolan~ such as co~cur-re~tly liquid stream has ~een reported by Gin/ et ~l., J.
Mi croerlçapsul a Li on 4; 3~ g-2 4 2 ( 1 9 ~ 7 ) .
It is furt~er envisioned that an undi~ferentiated cell ~hat is ~ oe~caps~lated ar ensapsu~ted with a 2S stabili~ing matrix, i.e., block or hollow ~i~ers, ~ill mature at a rate ~aster than that of a~ unenc~psulated cell.
"M~turation" as ~sed he~in, ~e~nq ~he ability of ~ cell to di~feren~ e or to ac~ie~e a specific biological ~ur.ction or ~et~bolic activity. TherefQre, a~ en~aps~la~ed ~ell ~ill ~0 ~ature at ~n in~re~sed r~e over a cell not so en~aps~late~.
Typically, the ma~uration rate will be at le~t a~o~ ~ 2.
fol~ ease. Si~ ly. when an in~rease in pro~iferation rate i$ desirable, t~e rate will be at least a~out a 2-fold inGrease. Not bein~ limited to a particular theory, it is believed that ~icroen~apsulatio~ prevents cell ~ggreg~tion .

0~17~ 10~ 13:32 FAZ~ 78 50~ FISH ~ RI~HARDSON P~C. I~bo22 the~e~y ~liminating cent~l necrosis of the ag~re~tes, 2n~
allows the enclose~ cells to grow ~nd mature duri~ culture.
~nother aspect o~ this invention is that encapsul~ion of ~
~ell also increases the cellls rate of grow~h ox prolifer~-S tion in culture. For exa~ple~ enclosing the neonatal isle~
~ells within a micro~apsule or stabilizing ~trix allows the~
to mature, grow, and diffe~qnti~te nto i~sulir.-~ecreting ~ cells during the in vitro culture stage as ~511 as after -mplantatiQn~ Furthe~, on~e the cell is encapsulate~ it oan lG ~e cu~red in vitro in either ~ serum-con~ainlng media or serum-free me~ia.
In a~other ~spect of the in~ention, more than one cell type can be co-en~psul~ted 1n the capsule. Thi~ includes ~oth na~ur~lly isolated or g~neti~ally m~nipulate~ cell~ ~s described herein. Cell ~ypes ~ay inclu~e distinct st, ges of a single lineage of cells ~e.~., bo~h pre~Ursor and differen-tiated end~crine cells~ o~ distinct cell lineage ~e.g., endccrine an~ blood) Therefore, ~he ~Ui~e of any cell type, incl~din~
n~on2tal, ~e~al, and~or pre-adult ce~, in such a protective growth-enhancin~ environment ~esults in a increased rate of maturation cr proliferation. Cell types in~lude endocrine ~ Gells, such as neuroendocrine and adrenal ce~ls: panCre~ti~
endrocine ~ells, endocrine precursor cells, such as stem cells and duct cells; hepatocytes, choli~ergi~ neurons, hematopoietic cells, hippo~z~mpa~ cells and the like. ~ther Gell t~pes th~k can be cultured within the m~ croGapsule or ma~rix in~l~de, but are not limite~ ~o, ~ny nesnatal, fe~al, pre~adolescent, adult, or other cell t~pe cap~ble of l~atura-tion and/or prcli~eratia~.
The followin~ exa~nples are intended to illustrate, ~ut rlot limit the invention. While t~ey are typical cf ~hose that might be used, other procedures ~nown to ~hose skilled in tl~e art m~y altern~ively ~3e used~

0~17~ MON 13: 3J FA~l ~la B78 50~ FISH & RICHARDSO~Y P.C. 1~lo23 2~ 7934 1 ~.x~rles.
The alms of ~he p~esent in~esti~ation were to develop a sta~dardi~ed method for the large sc~le isol~t~on of porGi~e neonatal isle~ cell ~NIC) aggregates, to define the cellular comp~sitio~ o~ these ag~regates, ~nd to assess ~heir growth po.entia~ and vi~ility ~oth in ~i~ro a~d J.~ vivo. The present invention shows the feasibility of usin~ Ihe neonatal por~ine p~ncre~s ~g ~ so~r~e of insulin-producing tissue for xenotransplantatlon into human Type I diabeti¢s.
~ased upon existing methads of isolating fe~al porcine isl~t tissue, a ~i~ple, relia~le pro~edure was developed for the pxeparation ~f porcine neonatal i~et cell a~regates with a reproduclble an~ ~efined ~elluLar co~posi-tion. Following 9 d~ys of i~ ro ~ulture, tissue from one necnatal pig pancxeas yiel~ed approximately 50,C00 ~sle~ celi aggregates, consisting ~f pri~ar~ly epithelial ~ells ~S7S~
and pancreatic endocrine cells (35%). Duri~g the su'ture period, t~.e total cell ~ass decreas~ initially, but su~se qu~ntly increased 1.5-fol~ between days 3 and 9. Tra~s-plant~ion of grafts consisting of 3 x 105 cell~ ~looo aggregates~ u~d~r the kidney ~apsule of allox~n-diabetic ~ude ~ice corrected hyper~lycemia in 75~ (10~13) cf the animals, whereas, 100% ~20~20) of recipients implahted wit~ ~ ~ 105 cells ~2000 aggregates) achieved eu~lycemia within 3 weeks 2S post-transplantation. Nephrec~omy of the graft bearing kidney at 14 weel~s posttr~nsplantation resulte~i in hypergly-cemi~ in ~ll recipients, and e~amination of the gr~fts reve~led ~he presen~e o~ nurnerous well-granulated insulin-and glucagon-contairling cells. The cellular insulin content of these gra~ts was 20 t~ 30-fold higher tharl at t~e t ~ne of transplanta~ion. Further, aggreg2te~ cultured in ~l~inate .~icrocapsules and the~ tra~splanted into diabetic re~ipients correcte~ the diabetes wit;nin 1-7 days post-tr~nsplantation.
~hese results i~dic~te th~ ~he neonatal porci~e pancreas c~
be used as ~ so~rce of iarge nu~bers of via~le i~let cells, ~ -13-0~17~ ION 13:33 FA2~ 78 50~ FISH & RICEURDSON P.C. 1~lo24 .
which have the pc~t~nt- a~ fo~ growth both in ~ o and in ~lVO, and exllibi.t the metal~olic cap~city to correct di~be~es.

O~f 17~ IION 13: ~ FA2~ 878 50~ FISH ~: RICHARDSON P. C. E~l 025 E~MPLEt MATERlALS AND M~ ~ OPS
Media a~d Produ~s.
The isolation of porcine NIC aggregates w~s ~ried out in Ha~k's balance~ sal~ solution ~5S, ~ibco, Burling~on, Ont,) supplemented with 0.~5~ bovine ~erum alb-~mir~ (BS~, fractlon V, Sigma, St. Louis, ~O), 10 ~mol/l HEPES (ICN
~iomedicals, Inc., Costa Mesa, CA), lOO U/ml peniciilin, and O.' mg/m~ streptcmycin. Ha~'s ~10 tissue culture medium was purchased from Gibco, isobutyime~hylx~n~hine (IB~) from I~N
Biome~ ls, nico~in~mide from B~H Biochemlc~l ~Poole, England~ and theoph~lline from Al~rich (Milw~kee, ~IS).
Insulin release experimen~s were ~arried out in RP~I tiss~e ~ulture ~e~i~m ~ co). Collage~ase ~Type ~! was ~tained from Si~ma, and crystalline trypsin, bovine pancreatic DNase, Pro~elnase K, and R~ase ~ from ~oehringer Mannhei~ (La~al, Quebec). Picogreen (P-7581), ~ ~luoxescent nucleic ~id s~ain for qua~tification of double-stran~ed ~NA was purc~ased from Molecular Probes (Eugene, OR).

Animals~
DorLor pan~rea~es were cbt2ined ~rom 1-3 day old Landrace-Yorkshire neonatal pigs ~1.5-2.0 kg body weight~ of either sex. Piglets were anesthetize~ with ~alothane and su~e-ted to laparoto~ ~nd ~o~plete e~sa~ginuation. The pancre~s w~s then carefully dissected from ~rrcunding tissue a~d placed in cooled (~~C) HBSS ~supplemented as a~ove~.
W~r~ and col~ hemi~ was ~ept to c10 ~n~ c5 min., respec-ti~ely. Eight gla~ds we~e initially used to st~n~ardize the procurement a~d isGlation pro~edure. Data were then o~tained ~om 10 ~onsecutive i~dependent expe~i~ents, with islet cells prep~red from 3 neo~at~1 pig pa~re~ses lor e~ch experi~ent.

~21-00~17~ YON 1~:34 FA~: Bl~ 878 50~ FISH 8~ RICE~R ~ 1 1q3 4 ~ 02~

. , .
-Male, inbredt ~thymic nude Bal~/c mice ~age ~-8 week~) were used as recipients of ~he NIC aggregates ~Jac~son La~oratorles, Bar Harbour, Ma~. ~ice were re~dered dia~etic by in~ra~enous ~njection of 90 mg~kg ~o~y wt alloxa~ (Sigm~, freshly dissolved in 1 mmol~l hydroc~loric acid~ ~ ~o 5 days be~ore transpla~tatio~. NormDglycemicr age-matched mice served ~s norm~l ~ontrols. ~ll xecipien~s en~erirlg ~his study exhi~ited bloo~ gl~cose le~els ~o~e 20 mmolJl. ~lood ~ples were obtained from the tail ~ei~ for gluçose ~s$ay (Medisense glucose mete~, Medisense C~n~a, M~ssiss~u~a, Ontario). ~n;m~l S were maintained under ~irus Anti~ody Free ~onditions in climatized rooms with ~ree access to sterilized tap w~ter and pellete~ f~od.

P.rep~ration ~n~ Culture ~f por~ine NIC ~ggregates.
Each of the glands ~ere cut into frag~e~ts of ~pproxi-ma~ely 1-2 ~m3, ~he~ transferred to s~e~ile tL~es ~ont~ini~g HBSS ~supplemented as above) with ~.5 ~g/ml colla~enase, ~d gently agitated for 1~-18 mi~. i~ a shaki~lg water ~ath 2t 37~C. The digest was filte~ed ~hrough a ~y~o~ scxeen (500 ~ 20 um), w~shed fo~r times i~ HBSS then place~ intc ba~teriolog-lcal pet~i dishes containing HAM's F10 tissue culture medium ~lO ~mol~l glucose, S0 ~ol/l IBMX, 0~5~ BSA, 2 mmol~l L-glutamine, 10 mmcl/l nicotin~mider }00 ~l peni~llin and 100 ug~ml strep~omyci~). Cult~re dishes were m~int~ined at 2$ 37DC (5~ C02, ~5% ~ir) in humidified a.i~ f~r ~ dayst with the medium ~n~ dishes ~a~ged the first day af~er isol~tion ~nd the medium e~ery seco~d day thereafter~
For some experime~ts, following ~ ~ys o~ cultu~e, approxima~eLy 50~ o~ the NIC aggrega~es from e~çh preparation were microe~capsulated with highly puri~ie~ algin~te ~eta-bolex, Inc.) producing u~ifo~m capsules of 250 ~o 350 ~m in diameter. Encapsula~ed ~n~ non-encapsulated ~ggre~ates were then further cultured for 8 dQys at 37bC i~ supplemented H~'s F10 containin~ 5~ ~v~v~ heat-activated autologous neo~atal pig se~um. ~o~rols cohsisted of non-e~capsul~te~
aggregates cultur~d fo~ 16 days ir.. serum free H~'s F10.

Characterization of islet cell pre~arations.
Following ~he i~olation p~oce~ure, and ~~ter 3 and ~
~ays of tissue c~lture, reco~er~ and purity of the NIC
aggrega~es was deter~.ir.ed ~n ~he ba~is of cellular h~rmone, ~, and ~mylase conteht. Al 1 ~easure.~.ent~ were a~sessed from duplicate aliq~ots of ~he islel cell suspension~.
Hor~one content ~as ~easured after ex4ra~on in 2 mm~
acetl~ acid cont~inin~ ~.25~ BSA. Samp~es were sonicated in acetic ~cid, centrifuse~ ~800 x g, 15 min.~, then super-natants w~re collecte~ and sto~ed at -20~ until assayed for insulin content by ELISA (Boeh.ringer M~nn~eim) and for glu~agon con~er.t by ~e~ns Oc radioimmun~2s~ay ~iagnostic ;5 Products Corp., Los ~ngeles, CA). .~myi~se cunte~t w~s deter~ined in supernatant fracti~ns ~ollected from cell su~pensions which were so~icated in supple~e~ted ~SS, stored at -20~C, ~hen ~e~sured by an en~yma~ic amyl~se assay (Be~km~r" C~rl.~bad, C~ or DN~ content, ali~uots ~ere washed in cit~ate b~$fer ~150 m~.o7~1 NaCl, 15 mmol~l citrate, 3 mmol~1 EDTA, p~ 7.4) an~ stored as ~e~l pellet~ at -20~C.
Prior to being assaye~, ceil pellets were placed in 4$0ul of lysic bu~fer (10 mmol/l Tris, 1 ~mol~ EDTA, 0.5t Triton X-100, 4~C, p~ 7.S~, so~ ted, supple~ented with ~5 ul of ~5 Proteinase ~ solu~ion ~8 mgl~l), vortexed, an~ incubated a~
65 and 70~C for 95 ~nd 10 min., respec~ively. Ly~tes were supple~ented wi~h Z5 ul of ~Nase A s~lutlon (10 mg~ml), ~o~texed, and inc~a~e~ for 1 h at 37~C. Alicuots of 25 ~nd 50 ul ~ere 2ssaye~ in dupli~ate by diluting them in 1 ml of DNA buffer ~10 ~ol/l Tris, 1 m~.oltl EDT~, pH 7.5) and me~suring fl~orescence at ~50 exr. ~ 51S e~. nm following the ad~itio~ of 1 ml o~ Pico Gr~en reagent ~1/2~0 dilutlon with D~A bu~fer1. S~mple.s w.re ru~ in ~aral~el with and diluted in propo~tion to a seven poi~t ~0 - 400 ng~,l) stand~r~ curve . n. r~ J~ r . ~

whlch was ~e~erated using calt~ thymus ~N~. In or~er to deLe ~nine DNA content per islet celL, NIC a~gregates were disso~iated i~ single cell suspensions ~y g~ntle agitation ~n cal.~i~m-f~ee ~ediu~ containing ~ypsin !15 u~ an~
D~ase ~4 u~ , xef. ~7-23). A Bu~ker cham;ber was used ~or cel~ ~o~n~s (27,2B), and sa~ples ranging from 2 - ~ x l~
cells were ~ssayed ~o~ ~MA co~tent.
T~e cell~lar co~positlon cf e~ch f~action was d~er~in.ed by elec~-on microscopy an~ Lmmunoh1stoGhemistry ~sing methods lC si~ilar to tAat prevlously described b~ Pip~leers et al (30-3~). Aggre~ates were rixed ~n t~ . 5~ ~v,~v~ ~lutaraldeh~e (Miilonig's buffer, ~ 7.2), post-fixed in 1.5~ ~wJ~1 Os04, washed in dist~lied water, t~en dehydrated successively in 50t 70, 80, ~0 and lO0~ ethanol, prior to e~be~ng in ~ral~ite, For elect~on ~icros~o~y, ~ectior.s we~e staine~
with lead citxate and ~-~hyl acetate ~hen subseque~tly exam7ned in a Hita~hi H 7000 transmission electron micro-s~ope. In e~ch sa~.ple, min~ y 100 cells were e~mi~ and c~aracteri~ed as exocrine (presence ~f zymogen granules ~50 n~ in di~,eter), endo~rine (presence of smalle~ gr~nules typical ~or a, b, d, or p~cr&a~i~ polypep~ide cells~, nongranulate~ serce OL se~etory ~e~ic~es), or as dam~ged kuptured ~lasma membranes and~or swollen organelle~) ~30,31~. ~o~ l~munohis~o'chemistr~, the a~idin-bis in complex ~ABC~ method ~25 ~se~ ~ith p~roxidase and dia~inobe~za~ine as the cnro~gen. Sections ~1 um) were a~fixed to glass sli~es ~y heat, th~ plast'c resin removed with sodiu~ methoxide a~d count~r stained with Harris~s hemot~xylin f~r 2 min., the~
su~seg~ently st~ined separately ~or the presence of insulin-~nd gll~cag~r~-containing ~ells. In each experiment, ~ min of lS aggrega~es randomly se~ecte~ fro~ ~ ~o 4 differen~
sectlons were ex~mine~. Primary an~ibod~es ~ako;
caxpinter~ A) included, guinea pig anti-porclne i~sulin ~l:lCoo~ a~d ra~bit anti-glucagon (1:100); biotinylate~
se~ondary an~i~odies and ~he ABC-enzyme co~plexe~ were ~2~

purchased ~ro~ V~tor L~borato~ies (Burling~e, CA~. P~i~ary antibodies were i~cubated for 30 min . (room te.~perature j, ~ile secondary antibo~ies were applied for 20 mi~.
or assessmor~t of i~ v~tro viability, the NI~s se~re-tory ~esponse to glucose w~s Geter~ined lollowing 5 days o~
~issu~ cu1ture by usin~ a stati,_ incub~tion a~say l27). The culture~ ~ractions were recovered ~ro~ the Petri ~ishes, washe~ and a~iq~ots o~ ~0 tc iO0 ~ggregates w~_e lnc~bated ~or 120 min. i~ l.S ~1 e f RP~ ~e~ium supple~e~ted witn 2 13 ~r.ol~' L-glut~ine, 0.5~ BSA and eit~er ~.~ mmol/1 glucose, 20 ~mol/1 ~lucose or ~0 mm.olil glucose ~lus 1O mmo~l thec~hyliine. Tlssue and m~dium were then sep~ra~ed by centriflg~tion a~d ~ss~yed ~or their respect~ve l~sulin cor~t6nts. T~e l~sul~n ~ontent of ~he medil~m wà~ expr~s~ed as lS a pe~en~e of the tot~1 content (i.e. tissue p'~s ~ediu~).
Stim~la~ion indices w~re ~alcula~ed by dividin~ the amc~.t o~
insul~n release a~ ~0 m~ol/l g~ucose ~+~- t~eophylline) ~y tnat rele~sed ~t 2.8 ~ol~1 glucose. T~ "our i~depende~t expor~ments a portior, of ~he freshly isolatPd NIC preparation was cui'u~e~ f~r 9 ~ays in the supple~ted ~AM's ~10 mediu~, but with~ut ~he ad~ition of 10 mmolJl nicotin~mide, in order to assess ~hether ~ otina~ide influenced ~he insu~in sec~etor~ ~ap~city ~f ~or~ine neonatai ~ ~el's.

Transplantation and metabclic follow-up.
Following 3 ~ays of culture, NIC a~grega~es were transplanted under the left kidney c~psu~ e of Halo~hane-ar.esthetized n~!~e ~lee. ~rior to implartati~n, the çellular composltion of the graft ~as ch~acterized as o~tli~.ed above, and in order to standardized the m~s~ of islet cells trahspl~nted in each experime~, represent~ti~e aliq~o~s o~ each preparation were coun~ed, sized, and the ~inal ouanti~y of aggregates was ~o~vert~d t~ tne n~ber ea~livalent to a di~eter o~ 150 ~m ~3~ uots c:onsis~ing of iOOQ o~ 2000 a~gregate equiv~lents were aspirated into -2s.

p~ly~thylene tubin$ ~PE-50~, pelleted ~y cer.~rifu~ation, an~
gently placed unde~ the kidr.ey capsul~ with the aid o~ a micro~ipul~tor syrin~e. ~nce the ~ubing w~5 remo.~ed, ~e ~psuiotomy ~as c~uterized ~ith a disposabl~ hi~-temper~ture cautery pen (A~ron Medical In~ustries, St. ?ete~sburg, FL.);
Tr~nspla~ted ~ice ar~d normal con~-ols were morlitored fo~
blood glu~ose l~els once a week ~etween B: 00 a~ 00 a.m.
W~en the ~lood alurose leve, was 5~.4 mm~l~l, the gra~t w~s ~ee~ed a succ~$s. At postt~nsplantation week 12, an o~al (O~T) ~n~ ther~ ~n intrape~itoneal ~IPGTT) g'ucose tole~ance test 48 h later, -~a~ perfor~.ed on ~rIC rec'pients with nsrmalized ~asal gly~emi~ an~ i~ ncrmal controls. Af~er a 2-h fas~ lL~ose ~3 ~gJg~ body weight! ~as a~nr.i~tered as a 50~ so'u~i~n ~ntr~sastricly or i~jected intra eriton~aly in~o n~n-anes~heti~ed mlce. ~lood samples were obtaired ~rom the tail veLn at 0,15, 30, ~G, ~nd 120 min~
~n ar.other se~ of experiments, ~ollcw7 ng 16 d2ys of culture, the ~lginate m~cro~apsule w~s rem~ovPd from some o~
the ~g~e~tes by incu~tiIlg the encap~lated ag~e~a~es in calci~ ee H3SS con~aining 0.5~ BSA an~ 1 mmolfL ethyle~e g~ycol-bis-~-amlno ethyl ether) N~Nl-tetra ~ce~i~ zcid for 30 min. ~Ton-en~a~slulated aggregates ~ere ~7sc treated in the same m~nner. Prior to implantation, in order to standardized the mass cf islet cell$ transpl~nted in ea~. experi~ent, repre~ent~ivP aliauots of ea~ preparatlon ~ere counted, sized, and t~e ~inal qua~tity of ~regates was ~onverted to the number equi~alen~ ~o a d~m~ter of lS0 ~m. Ali~uots of 2000 aggregate e~uiva~ents, both encaps~lated and non-encapsui~ted, were aspi~ated into polyethylene tubing tPE-3~ 50), pelle~e~ by ce~trifugation, and gen~ly plac~d ~de~ ~he ki~ey capsule o~ ~alothane-anQstheti~ed nude mice ~ith the aid of ~ ~.icreman~pulator syri~ge. O~ce ~he lu~ing was .e~lo~ed, the capsu~otomy was cauterized with a disposable ni~h-temperat~e cautery pen (Aaron ~edical Indus~ries, ~t.
~5 Pe.ers~urg, FL.). Transplanted ~ice were monitcrei for klood -26~

0~17~ 0~' 13:3S FA~ 78 50~ FISH 8~ RICHAR~S~ P.C. 42102~

gl~cose levels once a week be~een ~:00 an~ 00 am. When the blood g~ucose level was ~.4 mmol/lt ~he graft was deemed ~ success.

Cha~acteriza~ion o~ har~e~te~ NIC grafts.
At 14 weeks postt~a~splant~tion, NIC re~ipients undexwe~t a neph~ectcmy o~ the graft-bearl~g kidney for morphological a~al~is o~ to determine in~lin and glucagon contents of the har~ested grafts. The gra~+s in ~our re~ipients receivi~ 2000 aggreg~e~ we~-e however, no~
re~o~ed at this time, and these ~nim~l S were monito~ed ~o~ an ~itional 7 mo~ths . Neph~e~omized anim~ls were subsequently mcnitored to confirm a return of hyperglyce~ia.
The graft-~earing kidneys we~e immersed in Bouin's sol~ltion overnight and e~b~d~ed in paraffi~. Sections, 5 um thick, were then stained for the presen~e o~ insulin and glu~agon ~ontaining cells, ~s des~ribed above. Pie~es o~ native ~eonatal pig pancreas ~ere ~l$o processed an~ analyze~
accor~i~g to this procedure. In two re~ipient~, the graf~
and ad~acent kl~ney tissue was flxed L~ glutar~ldehyde t~en processed for ele~tr~n micro~cop~. For hormone extractio~, organs were homogenized and ~hen sonicated at 4cC in 10 ml of 2 ~mol/l a~eti~ a~ 10.2S~ BSA). Following 2-h at 4~C, tis.~ue homoge~ates were re-soniGated, centrifu~ed ~8000 x g, ~5 20 min.~, then supernat~nt~ were collected and the pe~lets further extracted by sonication in an additional 8 ml of acetic acid. The ~econd superna~an~ w~s collec~e~ after ce~trifugation, combined with the fir~t supernat~nt, tO~al volume was measured, and sam.ples were assayed for i~sul_n and gluc~gon content. The ~ me procedu~e was a~so used to extract hormones from pan~reases obtained fro~ NIC
recipient$, nor~l ~on~ro~ mice, and 1-3 ~y o_d neonatal pigs.

0~17/~ MON 13:35 FA2~ 78 50~ FISH & RICE!~SO~' P.C. E~102~
, Sta~istical Analysis.
~ta are exp~e~sed ~S me~rls i S~ of n inqeper:dent o~servatlons, St~tistical signific~n~e of ~ifferences was calculate~ with a two-taile~ unp~ire~l Student ' s t test ~ a one-way analysls o~ ~aria~ce ~ n case of multiple ~o~npa~isorls .

E~I.E 2 PO~C:INE ~TC PR~:PARATION
Following coll~genase dige~tion, a mean of 134 ug of insulin w~s recove~ed per neonatal ~?ancxeas ~T~ble I ) . The digest corltained approximately 65~ of the to~al irLslllin ~ontent preserlt in nondigested 1-3 day olcl neona~ ig p~ncreases, which were founcl to cont~in 205~7 ug insulin ~range = 1~1-242 ug). Tis~ue cult~e res~ted in further losses of ~el~ular insulin m~ss, so that after 3 ~nd ~ days of culture, 72 a~d ~4~ of the amou.nt initially preserLt in the digest was reco~rered, respe~ ely (Table I j . On the other han~, culture caused a marked reduc~tion in the amou~t o~
recoverable DNA, ~s less than 20~6 of ~h~t foutlc in the diges~
hras present after 3 ~ys of ~ulture and only 10~ following ~0 nine days ~pc0. 0001, Table I1 . Si~[~ilarly, c~ltured prepara-tions exhibited amyl~se valueis lower than 1~ of the digests (Table I~. The insulin content per micxogram of ~NA or pe~
amylase Gontent (U) o~ the porcine NICs signific:arltly in~re~sed during ~ulture ~T~ble I). On the b~sis of the ~S insulin per I~NA ~ralues, ~ day ~ltured NIC preparations were more than ~-fold ~}?C0 . 0001) enriched in erl~o~rine tis~ue comp~red to the ~reshly isolated m~teri~l (T~ble I ) . W~en exp~essed as a ratio of in~ulin per am~lase, enric:hment in en~ocrine tis~ue was r~ore tharl ~00-fold ~pc0. 0001) after the 9 d~y culture pe~iod ~Ti~ble I ) .
Ele~tron microgr~phs o~ the freshly ~sol~ted preFara-tions lndicated th~t ~g0~ of the ~ells were ncnenclocrine, ~f which the majority were exo~rine ~74~ and only 7~ were shown to cbnt~in secretory vesi~ es ~har~cteristi~ of en~ocrine 2~-VU~ lJ;.53 rA~ 78 50~ FlsH ~ RICE~ARDSOr~T P.C. 1j~02~

cells ~Table II~. Xmmunohisto~hemical analysis of the ~iges~
demonst~ated the presence of 5~ ~nsulin-positive and 2 glucagon-positi~e cells ~Table II). During the firs~ 2 days of culture, many degenerating cells were observe~ clea~ing away from the edges of a ~entrally lo~ated i$'et cell ~ggregate. Microscopically, NI~ ~ggregate~ ~eveloped into sphe~ical structures by the third da~ of culture and be~an to exhi~it a translucent ~ppear~nce similar to adult pan~reatic islets. Tissue culture markedly red~ed the percentage of exocri~e cells, so that at 5 days post-isola~ion, c5% o~ the cells were identified as exocrine (Table II~. These morpho-logical findln~s are therefore co~siste~t witn the obser~ed red~clion in amylase content followin~ cùl~ure. The nine day cultured prepaxa~ion~ consisted of ~5% stru~ur~lly ir~ t endocrine ~ells ~on~aining well-de~eloped en~opLas~i~
reticul~m tFi~. ~). This percentage of endo~rine cells was significa~tly higher ~han a the start o~ culture ~pcO~OO1;
T~ble II). I~ general, 9 ~ay cultured aggregates ~on~ained ~umerous no~grar.ulated epi~heli~1 cells ~57~) , as well a~
many duct-like structures were ~oUnc~ in the ~ggreg~tes ~Flg.
1). A low degree af ~ellular dama~e was als~ ol~serve~ in the electron micxogr~phs (Tab~e II). I~munohis~clogi~al e~amina-tion co~firme~ that 3 ~ay cultured preparations consis~e~
mainly of epithelial ~elis wi~h the presen~e o~ ~4%
ins~ cont~in1n~ an~ B% gl~cagon-co~taining cells scat~e~ed randomly throughout-the dggregate (Fig. 2c an~ 2d). This rar.~om distribution of end~crine cells wa~ also si~ilar ta tha~ observed ln the native n~onat~l porci~e pancreas ~F~g.
2~ and 2b~.
~0 The quantity of agg~egat~s re¢ove~d in e~ch experi~ent was estimated using the m~thod previously des~ri~e~ for determini~g human islet e~ivalents (331e The mean yiel~
obtaine~ from cne ~eonatal porcine pan~reas following collagenase digestion and 9 days culture was 48,~2~3125 aggreg~tes ~range - 28,~10-~0,966). Approxi~tely SO~ of the ., !

u~ ff FA2~ 78 50~ FISH & RICH~R~SO.~T P.C. E~oao aggrega~es measured between 5a a~d g9 um, 40~ we~e 100-14g u~ ~nd the remain~er were ei~her c50 um or r~nged ~rom 1~0-250 ~

E ~ T.F. 3 INSULIN SECRlTO~ RESPONSIVE~$8 The secxe~or~ a~ivity o~ NIC aggregate~ culture~ ,or days in the prese~ce or a~se~ce o~ 10 m~ol~ iço~i~amide was tested by co~pari~ the percenta~es of cellu~ insulin that ~as released at low glucose ~2.8 ~mol~l), hi~h glucose (~0 mmol/l) an~ high giu~o~e plus theophylline ~10 m~o~ o statistically significant dif~erences wére no~lced in the amounts of ins~lin secreted ~t the l~w glucose co~centration ~T~ble I~I). Incu~ation in 20 m~ol~l glucose signifi~ant~y (pC0.001~ i~crease~ ~he ~ecretory rate 3f bsth ni~inamide treated and nont~eated.~ ; this effect w~ further potenti-ated wher the NICs were exposed to high glucose plus theoph-ylline (Table III). Howeve~, the amount of insulin released and the ~alcula~ed ~tl~lation indiGes after i~cubation wi~h either 20 ~mol/l glucose or 20 ~mol~l ~lucose pl~s 10 mmo~l theophylline were significantly higher whçn the NICs were pre~ious;~ cultured wi~h ~icotin~mi~e. Nicotinamide tre~te~
a~greg~tes exhi~ited stimulation indi~es ~5-fold when ~omparing insulin release at hi~h glu~ose ~ersus that ~t low glucose. ~hen exposed to 20 m~ol/l glucose in comb.in~tion With 10 mmol/l theophylline, sti~ulation in~ices were >3~rold ~Table III~. An~lysi~ of the ~ot~l ce~lular insulin and DNA ~ontent reco~ered per NIC aggregate, a~d the percen~-age of _ns~lin-positi~e ~ells, revealed no significant differences be~ween nlcotinamide ~reated ~nd lm~reated preparations.

V~ O IIIUN l;S~ 78 50~ FISE & RIC~ARDSON P. C. [2 031 EXP~gPL~ 4 COMPO~IT~ON OF NIC GRAFTS
Prior to trarlsplantation, th~ composition of ~-da~
~ultured NIC grafts was ~e~enttirled by electroxl mi(;ro~copy, immlln~hi5tochemistry,, ho~one ~ontent, and DNA ~s~ay ~ L~41es II and IV). As p~eviously described, the NIC graf~s were co~posed of 57% nongranulated and 35~ endocr~ne ~ells; the remaini~g cell types.were i~ent~ied as e~the~ ~xocrine '3~) or damaged cells (5~; Table II). I~m~nohistoche~ical stainlrg ~or insulin and glu~2gcn was po~itive for 24 and 8%
of the total cell pop~lation, respectively (Tzble II). On the basis of the implants DNA content (Table ~ an~ the observ~tion ~hat single porcine neonatal islet ce~ls prep~red rrom 9-day cultured ~g~xega~es wexe found to cont~ir. 7.1 pg ~NA~cell (~ata not shown), it was calcul~ted that gr~ts cons~s~ing of 1000 or ~OO aggrega~es contained 1.3 or 2.
million celis, respec~ively~ In ~iew o~ the perce~tase o~
insulin-positive ~ells, the implants sho~l~ therefore ~o~t~in apprcxim~tely 0.3 o~ 0.6 ~x 106 insulin-produ~ing ~ ceils.
~C The me~surement of ~N~., insulin, and glucagGn co~ten~ also con~irmed th~t the mass of the ~000 aggregate grafts wa$
correspondingly l~rger than ~he 1000 a~greg~te grafts.

EXA~P~E S
TRANgPLANTATIQN OF NIC AÇGREG~T~S I~TO ~IA~ETI~ NUDE MICE
~fter allo~n ~inistra~ion, ali NIC reciplents e~hibited blood glucose le~els above Z0 mmolfl. ~i~oetic ~ontrols (ns~) not receivin~ a gr~ft were shoWn to survive fo~ 11+4 da~s. All animdls t~anspl~nted with 2000 N~C
aggrega~es exhibited ~iood glu~se values i8.4 m~olfl withir 8 weeks posttranspla~tation ~Table V~. This metabolic state W~ maintai~ed over the 14 week follow-up period, and in ~
a~;m~ls which were r.c~ ~acrificed at 14 weeks, normoglyce~.ia has been maintained for more than 11 months post--3.1 0~17/~ ~ON 13:~ FA~ ~la e78 509~ F~S~ ~ RIC~ARDSON P.C. 1~0s2 . .
transplantation. On the other h~nq, 10 of the 13 ani~als i~planted with 1000 aggrega~es achie~ed normoglycemi~ ~blood glucose ~8.4 ~mol/l) within the follow-up period (Table ~.
At 14 weeks po$ttransplan~atlon, blood gl~cose ~alues of those 1000 aggreg~te recipients not o~alning euglycemia were: 10.7r 1~.2r an~ ~4.4 ~mal~l. Compariso~ with nor~al controls indicated that recipients of 2003 NIC aggrega~es exhibite~ signiflc~ntly lo~er klood glucose level~ at h~ee~
1~ and 1~ (Ta~le V). These values d~d no~, however, i0 c~nt~ ue to decrease further, as those re~ipien~s ~n=4) ailowed to sur~ive long ~erm exhibited glucose levels of 5,2i0.~ at 11 months pos~transplantation (pc0.31 vs. normal controls). No diffexences were me~sured ~etween the me~r~
glucose ~alue~ o~ ~he 10 normogl~emic 100~-aggregate recipients and no~mal controls ~a~le V). n ~oth trans-planted groups, remc~al o~ thq gr~t-bea~ing kidney for morpholo~lcal exa~ination ~r hor~one extra~tlon was foLiowe~
by a rapid retu~n to the diabeti~state, indi~a~ing that the NIC grafts were responsible for ~he normoglycemi~ state.
~lucose tolerance tests were per~orme~ on norm~glycemic mice 12 weeks posttra~spl~ntation, and when compared to normal ~ont~ol ~ice, re~ipients of 2000-a~regates exhl~ited significantly lower giycemia values at ~11 ti~e poin~s (Fig.
3i. When the 1~00-aggxega~e recipients w~re co~ared to ~ormal con~rols, ~heir blood ~lu~ose levels were not s~tis-t~cally dif~erent throughout both tests. Co~parison of the two transplan~ groups indicated t~at ~he 1000-aggre~ate recipients ex~i~ited stati~tically higher valu~s at 15 a~d 30 ~in. i~ the OGT~ and at 15, 30, ~nd ~0 ~in. for the rPGTT
~Fig. 3~. In all grQUpS, the glycemia at ~in. 120 a~ter the ~olus of glu~ose was ~ot -i~niflcantly different fro~ the ~alues at ~in. 0.

0~17~ 31ON 13: 37 FA~ 78 50~ FIS~I & RICEARDSON P. C . [al 033 21 79~41 ~AMPr-~ 6 HORMONE CO~TF~T AND ~oR*R~tO~,~CAL C ~ ~ERIZAT~ON OF NIC
GRAFTS
Befo~e implanta~ion, NIC gra~ts containe~ on average 1.9 ug of insulin/1000 aggreg~tes or 4.0 ug o insulin/~000 aggregates ~Table I~. Fourteen weeks posttransplantation, conslderably large~ ~uantities of cellular insulin were re~overed fxo~ the graft beari~c kidneys tTable VI~. There was hGwever, no ~ifference in ~he gluc~go~ content between the harveste~ grafts and th~ gr~fts ~nalyzed a~ ~he time o~
imp~a~tation ~Ta~le ~ e insulin co~te~t of grafts obtalned fro~ n~rmoglycemic re~l~ient~ impianted with 1000 aggregates c~tained 30-fold (63.7 us~ ~ore insulin th~n what was initially transp~anted (T~ble VI,. ~raf~s retrie~ed fro~
animals receiving 1000 ag~regate~ and ~hich did not ~hieve eugly~emia, were show~ to Conta~in 12.~, 16.7, and 18.~ u~
lns~lin. In ani~als transpl~r.ted with ~000 ~g~regates, grafts cont~i~ed more than ~0-fol~ (88 u~ mo-e insuli~ than at the ~ime of implan~a~ion ~ble VI). The amount of insulin ext~acted ~om grafts o~t~ined from recipients of lO00 an~ 2000 aggregates corresponds to, respe~ti~ely, more than 74 and 141~ o~ the pancre~tic insulin ~o~ten~ in aqed-matched norm~l control mice (Table VI). ~lucagon content, ~n the other han~, wa simil~ to th~t ~ound i~
panc~eases of normai controls. . The recipients pancreatic insulin content w~s less than 1~ of that contained i~ normal cohtrol ~1mals, w~ereas thei~ glucagon con~ent ~a~ sl~ilar to that 1~ normal cGnt~ols ~Tabl~ VI~.
Macroscopically, cons~de~b_e growth o~ the NI~ gr~fts was e~ide~t ~ollowing 14 weeks p~transplant~ior.. ~muno-histological examinalion 4~ t~e grafts revealed a highly ~as~ula~ized tissue, ~onsistin~ predo~inantly cf well--granula~ed insulin- and glucagon-cont3ining cells (Fig. 2 e,g a~d 2 f,h xespectively).: Epithelial cells we~e ~ot f~equent1y seen i~ ~he ~fts. ~he 3 cells, whiçh composed 33~ , , . .. .

0~17~ lON 13:37 FAl~ 78 50~a FISEI & RICEIARI~SON P.C. b~lOS4 ~ 21 79341 the ma,o~ volume of the craft, were ~rranged in duct~
tub~lar-like stru¢tu~es ~rd the e~docrine non-~ ~ells ~ere scatte~ed randomly ~mon~st the ~ ~e~ No marked differences in ~orphology were observed b~twee~ the two tr~n~plant groups. I~ elec~ro~ ~.crogxaphs, do~or endocrlne cells were shown ta b~ ~tructurzll~ int~ct, hlghly granula~ed, and mitotic activ~ty was detected within some of the g~afts ~-cells lFig. 4 .

~YMPLE 7 lG MICRo~NCAPSUT~TION OF ISLET ~ELLS
Following g ~ays o' culture, NIC agg~egates were microenc~psul~ted with alg nate, as descriked i.n EXA~PLE 1.
~ Encapsulated aggreg~tes we~e cultured an ad~itional B days.
Some animals ~ere transpla~ted with encapsulate~ islets and lS some were transplanted ~fter removal of t~e capsule.
Tr~nsplante~ ~ice were mot-itored for blood glucose levels once a week.
Ta~le VII shows the _esults o~ transpl~nt~tion for 7 weeks post-tran~pl~ntatio~. Ml~e receivi~g cells cul~ured in the presence of the algi late rn~ c~ocapsule became n~rmo-gl~r~emi~ within 1-7 days of transpl~Ln~a~ion, while mice re~e~ ~ing cells cultured uLder non-e~capsul~ing conditio~s took nearly 7 wee~s to rec~ver to norn~glycemla.

SU~y The p~ese~t ~ta indica~e that ~ia~le porclne neo~atal i~let cells can be successf~lly lsol~ted in l~rge n~bers by ~ulturing co~lagenase digested panc-e~s ~or 5 days. Sin~e w~
and others ~34) obser~ed taat 't~e e~ocrine portion of the neonatGl pig pancre~s doer not. contain inta~t and m~tuxe istets, ~ut rather exh;~;ts a r~ndom distribution of endocrine cells, no attem~ts were ~a~e to process these organs usin~ methods convention~lly used for isol~ing adult ...... _ . 1.'.

0~17~0 NON 1~: 3~ FA2~ 78 50~ FISH B; RICH.~R~SO~ P.C. E~1035 ~mmalian islets. Neo~atal pig pancreases were therefore, digested a~cor~ing to a modifi~ation of t~e method cf Korsgren et ~i ~lO} for prepaxing fet~l pig isiet cell cl~sters. Tis~ue ~ult~re was then use~ to enrich the pr~parations in e~docri~e cells prior to assessing thei~
~iability hrou~h an in vitro sti~lation as~ay and by transplantation into diabetic ~u~e ~ice . Sele~ti~n of the medi~ supplements was based on the followi~ ~o~si~erations:
l) se~Um was omit~ed ~ue ~o its ability to pro~ote the sur~ival o~ ~ontaminating fibro~lasts a~ pancre~tlc exocrine ~ells, and based o~ previouS repor~s (35,36~, albumin ~as selecte~ ~s a se~um-substitute; 2) IBMX was used for its capa~ity ~o stimulate DNA synthesis i~ rat isleis ~37), a~d its p~tential to enha~ce sur~ival of c~itured ra~ ~ cells 136); 3) lO m~ol~L gl~cose was a~ed to enh~ce ~ ~ell repllcation (38, 3~) ~nd for its cytoprotecti~e effe~t during culture o~ p~rifie~ rat ,~ ~ells ~36) i arld 4~ ~ico~ina~r.ide was inclu~ed due to its cbillty to sti~nulate islet ~ell DNA
replication ~40~, ~nd its ~e~eficial effec~ on the meta~oli~
functian of porcine feta~ islet cells ~l2).
Using insuLin content as a paxamete~, approximately 6S%
of t~e inltiai pancreatic ~ ~ell m~ss was re~overecl a~ter ~he ~o~.la~e~ase di~estio~ ph~se. This in~e~ of ~ coll reco~ery is llkely an u~der e~ti~nation because it does not ~ake into account possi~le degranulatio~ wh7ch could occu~ duri~g the dige.~tion. A nine day culture of ~}le digest suc~eed~d in ellminating Ihe m~jority o~ conta~ina~ing exoc~ine cells an~
~esulted in the ~or~ation Or numerous i~let cell a~regates.
The NIC aggrega~es were predo~nantly co~posed of epithe~ial cell~, e~hibited ~ puri~y of 35~ endocrine c~lls, and contained appxoximately 2S% insulin-positive cells. ~ith the technique used in thls study, ~ene~ally 50,000 NIC aggregates were reçovered from one panc~eas. These yields ~e signifi~antly higher th~n those obt~ined in studies using ~id-gestation~l fetal pi~sr where the avera~e ~ield per fetus ~35-0~17~ [ON 13:38 FA~ 78 50~ FISH & RIt;HARDSON P.C. 1~lo3~

. I
w~s approximately 10,000 lslet cell clus~ers ~10,133~ ~he~
considering the me~n ~NA ~e~ove~y per p~n~re~s, the DNA
content o~ porcihe neonatai islQt ~ell~ ~7.1 p~!cell), and the pex~ent~ge of ~.nsuli~-p~sitive cells, the following ecuation can be used to calcu_~te total ~-cell m~s in the isolated NIC ag~regates:

Total ~NA content X % ;~ lin ~ositive cells 7.1 pg ~NA~NI~ 100 =number of ~-cells recovere~ pe~ pancre~s ~he nu~ber of 3 cell~ recovere~ from f~eshly isolate~;
3-day, ~n~ 9-day ~ultured p~ep~ratio~s i~ ~alcul~ted to be ~ , 8~5, 13.3 million cells per pan~reas, ~espectivel~.
Si~il~ly, when considexing ~he percentage of ~-cells the calcul~ed nu~ber of alpha cells is 11.7, 3.2, an~ 4.4 millio~ eells per pa~cre~s, ~espe~ti~ely. ~he decrease in endocri~e ceLl mass 4e~ween the isolatiDn an~ 3 d~s culture is likely the result of celeteriou~ effects of the collagenase digestion and the presence of potentially cy~otoxi~ proteases release~ from degene~ating exo~rine cells during ~lltu~e. ,n can~rast, the increase in both ~ and ~
cell mass ~i,e. 3~ and 5~, re-pecti~ely~ between day 3 ~nd day g of culture, can possibly be expl~ined by the g~owth or differentiatior. of new en~o~ri~e cells. Cellular in~ulin content also decreased signifi~.ntl~ ~uring the ~irst 3 ~ays of cul~ure, and this is pre um.bly relate~ to the fall in celi mass. ~owe~er, ~etwee~ days 3 an~ 3 o~ c~l~ure, cellular i~sulin conten~ decre~sed by lo~ et ~ ~ell m~ss w~s shown to incre~se. Thus, ~ven thoug~ new 3 ~el~s were 3C forming an~ ~ontri~uti~g to the total insulin poo', their ir.~uli~ sto~es as ~ell as t~at of pre-existing ~ ~ells likely :

O~J17/~ MO~ 13:3~ F.~ ~ 78 50~ FISH ~ RI~HARDSON P.C. I~bo3~
2 1 793~ 1 ~ecreased as a result of secreto~y acti~i~y ~hat ex~eed~ the rate of insuli~ bio~ynthesis du~ing ~ultu~e. The ~ecove~y o~
cellular DNA de~reased thro~hout the 3-da~ culture period, whi~h is likely the result of the m~ked eli~ination of cont~min~ g exocrine cells.
Many studies h~ve indicated ~hat ~he felal ~ cell has a poor Insulin respo~se to gl~çose, w~ich is rapidly converted to a ~ore ~dult pattern ~fter ~irth (22-25~. In th~ prese~t study, porcine neonatal ~ ~ell~ were cap~ble of se~reting sig~lfi~nt amounts of insuli~ in respon~e to a ~lucose challe~ge. This secretory capacity was further augmented when ~he NIC aggregates were p~e-cult~re~ in the presen~e of nicotinamide. T~ese result~ are eom~arable to those described for islet cells prepa~ed fxom the neonatal pig panc~eas (1-3 d~ys old) ~fter trypsin ~isso~iation ~n~ 7 days culture ~27), and are sig~ificantly higher th~n those observed for fetal pig islet cells ~10, 12); suggesting that in the pig, neo~at~l ~ cell~ ~re more ~e~p~nsive ta glucose ~h~2l fet~1 ,e cells. While no~ wanting to be bourld b~ a particular th~or~, ~ possible explan~tio~ ~or ~icotinamide's beneficial effect is th~t it may ~ct as a p~otectlve agent, thereby preserving the insulin s~cre~ory ac~ivity of the neonatai ~ cell th~oughout the.cultuxe period. Nicotin~mide has ~een shown to protect against the cy~otoxic action cf s-treptozot~ci~ on ~ cells ~;41,;4~) and ~o slow or arrest the development of diabe~es in ~he non-obese di~betic ~ice (43).
The agen~ may ~herefore enhance survival of por~i~e NICs after ~ollagenase digestion an~ p~otect ~hem ~rom ~hemical disr~ption ~ue to the pro~eolytlc acti~i'y of e~zymes leaking from degenerati~g exocrine cells during cultu~e.
Interes~i~gly, ~ulture in nicdtin~mide did not incre~se t~e freque~cy o~ insulin-positive ~ells in the NIC aggre~es.
This obser~ation is in ~onLra~~ to that obser~e~ when fet~l porcine ~lZ) and h~man ~44)'isl'e~ ~ell cl~ster~ were cultu~ed with nicotinamide.
~7-, 1, 0~17i~ hlON 1~:39 FA~ ~la ~78 S0~ FIS~ RICEI~RDSON P.C. E~1038 ~iabe~ic nude ~ice were t~ansplanted wit~ 1000 or 2000 ~IC aggregates, cont~inin~g either ~ or ~ x 105 ~ cells. ~t posttransplantatio~ week 14, 75~ of the ~ice receiving lCiOo agg~egates exhibi~ed blood glu~ose values +8.4 ~ol~l, whereas, ~000 ~ggregates cured di~betes in '00~ of the animals within 8 weeks ~fter implantation. In ¢or~trast, Da~ilii and asso~iates de~onstrated that 2~00 a~ult po~cine i.slet equivalents i~frequently produoed ncrmogly~emi.a in diabetic nude ~i~e, ~nd t.~at 400C i~et e~ui~alents with ~n a~era~e celiular lnsulin ~PSs of 38,4 ug ~equlred ~5 ~eeks to normalize dl~betic nude ~ice (15). In the pre3ent study~
only ~OGO pcrcine ~I~ aggr~ates containing 4 ug in~ulin, wa~
~equired to achieve eu~lyce~ia long ter~. One possi~le explanation for these differences is that ad~lt porsine lS isle~s axe considerably ~o_e fragile than the ~IC aggrega~es and thi~ may ~esult in significantly more ~ c~ll dea~h and malfun~tion of the a~u~t _slets pos~transpl~htation. It i~
also c~nceivable that during the initi~l days after i~plan~ation, reduction ir the grafts ~ cell m~ss may occu~
due to poor vas~ulari~ation and anoxic conditions. ~hus, with adult islet grafts t~e flnal ~ cell ~ass that becomes functionall~ ac~ive fol~owin~ the engraftme~ perio~ ~ay be considerably less rhan th~ amount initiall~ implanted. On the other har~d, the ablli~y of imm~re NICs t~ differ~ntiate a~ prolifera~e may pote~tizlly allow them tc replace an~ ~
cell ~ass lost ~o ische~ damage i~ the i~mediate post-transplantaticn period. ~lth~ugh the N~C grafts grown in ~he absen~e of cult~re in m~crocapsules were unable to ~orrect di~betes immedlately a~ter tran~plantatio~, the~
event~ally developed the capscity to establi~h and main~in eugiyçe~ia, likely be~2u~e the relatively .'ew ~-cells implanted i~itially were ~ubse~uently supplemented by the growth ~nd~or differe~ti~tlon of a~ition21 new ~ ~ells.
Interestingly, all recipi nt~ survived ~his hyper~lycemi~
period, ye~ betic ~o~t~ols survive~ for only 1114 d~ys -38~

,, O~f'17Ja~ I~ON ~: 3a FAll ûl~ ~78 509~ FISH & RICE~R~SON P. C. 1~103~

afte~ alloxan treatment, sug~esting tha~ ev~n in t~e ~irst two weeks posttr~nsplantation, NIC gra~ts pr~d~ced suf~icient ins~lin to keep recipien~s ali~e, ~lthough no~ e~glycemi~.
~e hypot~esi~e that ~ hype~glyce~ic e~vir~nmen~ may be essential to ~nducing the growth an~ differentiation of new ~-~ells in our e~peri~ental ~odel, and perhaps also i~ the clinical setting. ~t the timd ~ impla~tation, the insul~n ~ontent of NIC grafts corres~onded to only 5 - 1~% of the p~ncreati~ lnsuli~ content ~ound in age~ matched nor~al con~ols. Following transplantation, the grafts i~sulin ~ass incre~sed by ~20-fold. WheLher this increase in insulin m~ss w~s rel~ed to the birth of new B cells through diffe~entiatio~ of epithelia1 cell~ in the ~T~C aggre~ate~
a~d/or replication of exis~ing ~ ~ells w~s ~ot assesse~ in ;5 this s~dy. It ha~ however, been suggeste~ that the m~jor sou~ce of newly formed ~ cells;i~ ~ranspl~nted por~ine islet cell ~lusters is fro~ undiffe~enti~ted ep~ theli~l cells rathex than from p~e-existing ~ cells (11). Our ~orphologiGal data indirectly suppoxt this concept, ~inse at Lhe time ~f lmplantatlon ~IC grafts wexe ~o~pose~
p~edom1 h~tely of epitheli'al ~ells, whereas se~eral weeks afte~ transplantation, few ~epi~heli~l ~ell~ were d~ected an~
insulin-producing ~ cells now co~prise~ the m~jor volume of the ~raft. It ~nnot ~e ex~luded, however, th~t ~ ~ell proliferation ~id not ~ontri~ute to at lea~t some of the ir.creased i~sulin ~ontent of the gr~fts~ ~s ele~tron micrographs i~dic~ted mitotic acti~i~y within some Qf the engrafted ~ cells. The usn of semi-q~a~t~t~ive morp~ometric ~echni~ues, such as ~!romoldeaxyuridine l~beling and si~ultaneo~s im~unostainirls folr isiet ho~one~ tl~), should provide furthe~ insi~ht irto the growth kinetic~ of porcine NIC qrafts. ~t is worthy to note th~t the ~-aft~ glucagon content did not inorease following transplan~a~ion, ~ggesting that ~o ~dditi4nal 5rowth of ~-~ells a~urred;
Simil~rly, in s~udies wh~re fPtal porci~e islet ce~ls we~e ~39-0~17J~ b[ON 1~: 40 FA~ 78 50~ FIS~I & RIGHARDSON P. C, E~l 040 2 l 793~ ~

~mplanted in~ allo~an-di~beti~ e mice, t~e fre~uency of glucagon containing cells markedly clecreased zfter transplantation ~ . These obse~vatiorls indica~e th~t in ~rrl~nature islet ~ell grafts, continued gro~th an~
di~ferentiation of endo~rine non-~ cells is limited, i f nc~t inhibited, when ~r~nspl~nted under the kidnçy ~apsul e of allcxan-~iabetic nud~ ~nice.
However, neonatal i~let ~ells cultured in algin~te micro~apsules for ~pproximately 1 week, likely ~ontaine~
increased ~-cell mass. There~o~e, o~ transplanted into the reoipient, animals became no~mogly~emic in one to seven ~ays.
It is interes~ing t~ no~e th~t once norm~glyce~r,ia is a-chieved, it appe~rs that the transpianted cells no longer prol i f er~te The pancre~s of normal control mi~;e w~s shohm to contai~
a~out 40 ~g insulin and grafts o~tained fr~m re~ipienl:s of 2COO NIC agg~egates ~ontained 8~ u~ ir~sulin. The ap~arent exçess ~ ~eli ~ass i~ these Anim~ls co.lld explain their lower blood glucose levels when ~ompared to age-m~tched nor~al ~O controls. Or. the ~ther h~nd, this phe~o~enon has also been observed in nude mice ~ransplan~ed wi~h adult porcine islets in which the grafts ~ ~ell mas~ did ~ot diffe~ fro~ th~t fou~d in the pancreas of nor~al control mice ~15). Thus, an alternative escplanation for thi~ obse~vation is that t~e ~5 donors ~ ceils e~entually regulated the re~ipient ' ~ glucose homeostasis to that found ir~ pigs, r~Lthex than i~ ~nice.
Therefore, since plasm~ glucose . levels i~ pigs and h~n~ns are slr~ilar, these results indic~te ~at tr~nsplantatlon o~
porcine NI~s into hur~ar~s ~houl~ theoretically mai~tain blo~d sugars within ~he ~e~ipients physi~ogic~ ~ange.
rn conclusion, the neon~l porcine parL~reas c~n be u~ed for the isol~tio~ o:~ a large nurnber of functionally ~iable islet cells. E'urtherrrlore, due to the~ r ready av~ilability arld inhe~ent c~pacity to proliferate and differentiate both 3S in vi tro and in vivo, ~hey constitute arl at~ractive so~rce o~
4~
i 0B~lT~ 3[ON 1~: 40 FA~ Bl~ ~78 50~ FIS~ ~ RICHAR~SON P. C. [~!1041 2 ~ 7~34 ~

insulin-produ~ g tissue for r~t.udies of islet cell neogenesis or as ~ source of ~enogena ic islet cell5 for clinic~l tr~nsplarltation . . ' i.

Table 1 Prepardtion of porcine neon~t~l islet cell aggregates R~n~ perP~ncleds Pur~y ,p Culture l~enod Insulin (~lg~ ~NA ~llg) Amyhse ~ ~nsul~ lgp'DNA ~ nsu~ g~4mylase ~U) 'D

Freshly isolate~ 1~4 + ] 2 414~ ~t 464 60.03 + 5.40 0 032 ~ 0.002 2.2 ~ 0.
~l~ay~ gk+7~ 7~3~ 0.42~0.01~ Ol2g+0010~ 222.1~16.0 9Days 86*~t 3g4+24~- 0~04+0001t Or215~0~011~ 2I~1.1~77.
.. . . . . .
~ ,, u -- -~~ ~ V~es are me~s + ~ ~~e~ent ex n nts ~ e~h ~e~ por~e ~C a~d s ~re ~p~d ~ 3 p StatistiGall ;fi~n~,~eof~ nu~wasc~lculatedbyone-wayanalysisofY~ance n *p~0 05, ~pco ~ p<0 0~01 vs. ~e$hly i~olqte~l ~)~
~p<O.OOOI vs. ~ Day~. l ~2-OB~17~B MON 13:41 FA~ Bl~ B78 50~ FIS~ & RICHAROSON P.C. 1~043 2 ~ 7934 1 t ~

g.

E

c e ~ ~
8 ~ Z ~ ~ ~ ~ ~ ~ ~

e ) ~ 8 8 o 3~ o o 0~l7~ ON 13:41 FA~ el~ e78 50~ FISE & RICHARDSON P.C. l~1044 2 1 7q34 1 q c ~ r o a 3 y y y 3 o ~ ~ ~ + + . 8 y 3~ C~

~ o ~ o 3 ;~ i 9Y i~

, ~1 Table lV. ~ ,; Q~ n of porcine nPI .t~ islet cell ~ra$s prior t~ tl a~u~ol lation.
Content (,L~gJgr~) Puri~ ,Ug~ ~

Gra~ Insulin Glucagon DNA Il~sulmf~NA G1uc~gnn~DNA ~P

1000 NIC ~b.~&t~s 1~0.3 O.G2~0.0~ 8.g~0.7 0 21~0.022 0.073~0.008 2000 MC ~ 4t~ 4 0~0 4 1.3~0.11 18.~1.~ 0.21g~0 02~ 0.072~ 007 co Yalues are means ~ SE of lQ jn~ en~ exp~Lw~ each ~ e ;~ r~ine NIC aggrc~ates were ~r~aled f~om 3 panc~ea~e~.

... . _ . ................................................................ x __ OB~17/~ h[ON 13: 42 FA~ 78 50~ FISII B~ RICHARDSON P. C. 1~!104~
21 7934 ~

.

~~ ~ ~ , L

,L

X ~ _ _ ? Ct~ -C ~

-- 2 L V!
~ ~ 3 ~
$

~, ~ ~ o .~ o e ~ ~ o o , o 8 . 13 ~3 c9 U~ o , U~
, 0~17,'~ IIION 1~:42FAZ~ ô78 50~ FISH &~ RICNAR~SON P.C. l~1047 . ~ , J ~

m ~ I ~ g v a ~ o~ oo ~ ~ ¦ 7 u o a c ~

- ~ ~,a~

4. . O U~

0~17~ 1110~ 1a:42 FA~ 78 50~9 FISH ~ RIC~SO~ P.C. ~ 4~

.
~o ~R ~ o 5 e 7~ ~ 7 !: ~ 5 0 o o ~ ,~, q ~ ~ ", 5 ~ ~ ~ ~o _ ~ a ~

5 ~ ~ 5.~ ~' 5 ~' ~ 5 y E

E I y ~ ~ 5 ~ Y ~E b Ul O

.... .. ..... ..... . .

0~17J~ ~ION 1~: 43 FAl~ 78 50~ FISH B: RICHAR~ON P. C. Iel 04~

~E FER~NCES

1. Scharp, ~.W., P.E. L~cy, J.~. San~iago, C. S . ~c-Cuilough, L.G~ ~eide, L. Falqui, P. Marchetti, ~.L. ~ g-eri~h, A.S. Jaffe, P.E. Cryer, C.B. An~erson, and M.W. Flye.
19~0. I~su~in inclepen~n~e ~.f~er isle~ transplantatio~L into type 1 di~etic patient. Diabetes 3g: 515-S1~ .
2. Warllock, ~.L., N.~. ~e~man, E:. Ryan, ~.E.A. Seelis, A.
Ra}~inovitch, and ~ ot;t~ 19~ orrrLogl~cert~ia after transplantatio~ o~ freshly isolated a~d ~yopreservec~
pancre~tic islets in type: (insulirl-dependent) diabetes mellitus . r)iabetolo~ia 34j: 55-5B.
~. Socci, C., L. F~lqui, ~.M. Davalli, ~. Ricor~i, S.
Braghi, F. Bertu~7i, P. N._ifi, A. Secchi, F. Gav~2zi, M.
~reschi, P. Magistre~ti, S. occi, A. ~ignali, V. I:i C~rlo, and ~. ~ozza. 19~1. Fres~. human isler t~nsp~ a~ation to replace pancreatic endocrin~ fu~ction in type 1 cIi~betic pa~ients . Acta Dia~etol 28 :151-157 .
4 . Warnock, G. L ., N.~ . Kn~teman, E . ~yan, A. Rabirlovitc~, ~d R.V. ~ajotte. ~g92. Lon~-term follow-up ~fter tr~nsplantation of i~sulin-~roducing pancreatic islets into p~tie~ts with type 1 (insul'r-dependen~ dlabetes mellitus.
~iabe~olcgia 35:~-g5.
5. ~icordi, Ç., A.~. T2aki-, P.B. Ç~rroil, Y. Zeng, H.L.
~odriguez Rilo, ~. Alejandro, ~. Shapiro, J.~. Fung, A.J.
~emetris, D.H. Min~z, and T.,~ St~21. l~g2. Human islet isol~tion an~ allotxan~plan ~tio~ in 2~ consecutive ca~es.
T~ansp~a~tation 53:~07-414.

.

0~/17/~0 llfON 1~:43 FA~ ô78 509~ FIS}I & RICEIARDSON P~ s21050 .

6. Al~ dro, R., 5. S~as-er, V~ Es~u~nazi, ~. B~rke, J.
Nev~y, J. Miller, E.T. Shapi~o, C. Ri~ordi, ~r,~ int~ .
1994. Long-term survival o, ~ntrapo~tal islet al3.og~ts in type 1 ~l~betes mellitus. Ir Pancrea~ic Islet Cel~ Tra~s-plan~ation. ~. Ricor~i edl.tor. R~ Lan es CG., A~stLn.
410-413, ii.

7. Gray, ~.W.R., P. McSh~e, ~. Grant, ~nd P.J~ ~orris~
1~84. A me~hod for isolatio~ of islets o~ ~an~e~hans from ~he hu~an pancreas. ~iabetes 33;1055-1061.

8. War~ock, G.L., ~.~. El~i8, R.Y. ~a,otte, l. 3awidson, S.
Baekkeskov, and J. Egebjerg.~ Stud~es o~ tne isolatio~ and ~ia~ility of h~man isl2ts of ~.ngerhans. ig~8. T~nspl~n~a-tion 45:957-963, 9. ~icor~ ., P.E. ~acy, ~.H. Fi~ke, B.J. Ol~ck, ~nd D.~.
Scharp. 1~8~. Au~omated ~e~hod for isolation of huma~
p~ncreati~ islets. ~ia~etes 37:413-420.
LO. Korsgren, o., S. S~dle~, A,S. LandstrUm, ~. ~ansson, znd ~. Andexsso~. 19~. Large-$~ale produ~tion of fe~al por~ine ~anereatic isletlike ~-11 clusters. ~n experi~ent~1 tool for studies cf islet ~ell ~i~feren~ia~ion an~ ~eno-transp~antation. Transplan~-ion 4~:509-514.
11. ~orsgren, O., L. Jan~son, ~. Ei~irik, ~nd A. Andersson.
l9gl. Functlon~l an~ ~orpholo~ical di~ferentiation of fetal porcine is1et-like clusters ~-ter transplanta~io~ into nu~e mi~e. Dia~et~logia 34:37~-38j.
12. Korsgren, O., A. ArLde~son, ~nd c. S~ncl~r. 19~3.
Pxetreatment of fetal porci~e pa~cre~s in cu~ture with nicotl~amide accelerate~ reve~sal a~ ~iabetes a~ter tra~s-pl~ntation to nude mice ~ Sur -ery 113:205-214.

i O~J17~ WN lJ: 43 FA~ 78 50~ FISH ~ RICH.4RDSON P.C. 4~1051 13. Groth, C.G., O. Korsgr :1, A. Tibell, J. T~llem~r, E.
M~iler, ~ Bollnder, J. Ostnan, F,.P. Rei~hoi~, C. ~eller-str~m, and A. Anders~on. 19''4. T_ansplantatio~ of porcine ~etal p~ncre~ to d~abeti~ p~tie~t~. La~cet 34~;1402-1404.
14. Liu, X., K.F. Fedexlin,~R.5. Bretzel, B.J. Hari~g, and M.D. Brendal. lg~. Per~ tent reversal of dlabetes ~y transplan~tion of fet~1 p g prcislets into ~ude mi~e.
Dia~etes 40:858-~6~.
15. ~av~lli, A.~., Y. Oga~-a~ ~. Scaglia, Y. ~u, J. ~olli-s~er, S. Bonner-Weir, and G.~. Wei~ 95. Fun~tion, mass.
and replication ol porcine ~nd rat t slets ~ra~splanted in~o diabeti~ nude miCe. Di~bet~s 44:104-111.
~ archet~i, P., R. Gianna_elli, 5. Cosimi, P. Masiel~o, A. coppelli, P. Viaca~a, and R. ~ valesi. 1~95. Massive isol~tion, morp~ologi~al and fun~tio~l char~cteriza~ion, an~
xenot~ans plantation of bo~_n- p~nc~eatic islets. ~iabetes 44:375-331-17. Ri~ordi, C., C. SoC~i, k.M. ~a~alli, C. St~dacher, P.
B~ro, A. ~e~tova, I. Sassi, F. Gavaz~i, G. Poz~a, and V. ~i Carlo. 1~. Isolation of t;:e elu ive pi~ islet. Sur~ery 107:688-694.
18. Socci, C., ~. Rico~di, A.M~ Davalli, C. Staud~cher, P.
Baro, A. Ver~ova, M. ~resch'., F. Gav~zzi, S. Braghi, G.
Pozza, arld V. Di Carlo. 1989. Se~ection of donors si~nifi-c~ntly impro~es pig islet tsol~tlon ~ield. Hor~n ~etab Res ~5 IS~ppl. 1):32-35.
lg. Kirchh~f, N., B.,J, Her~ng, V. Geiss, K. Federlinr and R.G. Bretzel. 1394. E~ide~ce for breed-dependent ~if~er-eIlces in porcine i~lets of .IJangerhans. Tran.splant Pro~
26 ~16-~17.

;

.. . .. . . .. .

0~17~B IION 13: 44 FAZl Rl~ ~78 50~ FISH B: RICHAR~SON P. C. Iib 052 ;
20. V~n 3el~ne~, J.H,M., ~.E. Hulstaert, ~.H.J. W~l~ers, ~n~
~, va~ S~hilfgaarde. i3~2, SignifiGan~e o~ the peri--insulinc~r extracellular ~atr-.~ for isl~t i50l~tior, ~rom the pancreas of the ra~, dog, p i~, a~d man. Cell ~issue ~es 267:13g-14~.
21. Marchett~, ~., E.~. ~inke, ~. Swa~son, ~. C-er~
si~idi-Vazeou, D.W. Scharp, R. Navalesi, and P.E. ~acy.
13~2. The pote~tial of porci~e islet xenotra~sp~antation i.n ~he the~apy of diabe~es. . :i~b Nutr Met~b 5 (Suppl. 1-):151-154.
2~, A~plund, ~., S. Westma~, and ~. HellerstrlJm~ lg69.
Gluçose stimulation of insul n secretion from the isolated panc~eas of foetal an~ new bor~ rats. Dia~etologia 5-:~60-2~2.
23. Asplund G.... ~ynami~s of 'nsuli~ ~elease fro~ the foet~l ~nd neonatal rat p~nc~eas. . .~3. Europ. J~ Clin. In~est.
3:338-344.
24. ~hoten W,B.. 1930. In~_Lin secretory dy~amic~ during de~elopment of rat p~ncreas. ~ J ~hysiol ~3g:E57-E63.
25. Hole, ~.L., M.~,~. Pi~n-C~ith, a~d G.W.G. Sharp. 1~88.
Developme~t of the biphasi~ re,~o~se to glucose ln fetal a~d neonatal ra~ pancreas. Am J ~hy~iol 254:E1~7-~174.
2~. Tuch, B.~,, R. Jones, and J.~. ~u~le. lg~5.
~turatio~ of the respo~se of h~..~an fetal pa~creati~
e~pl~rlts. Diabet~logia 2B:2:8 ~1.
27. Korbutt, ~.S., and D.G, ~.peleerq~ 1~9~. Çold sto~age of the r~t pan~rea~ ~efore ~ ation ~f islet ~-c~lls.
Dlabetes 41:~5-307.
28. Pipeleers, ~.~., P.~. Ir t Veld, M. Van De Win~el, E.
Maes, ~.C. S~huit, ~nd W. Gept . lg85. ~ new in ~ltro ~odel fo~ the study o~ pa~creatic . a~d B ~ells. E~doc~i~ology 117~806-81 .. .

., :
~''. ' .

, 00~17,'~ MON 1~:44 FA~ 7~ 50~ FISH & RICHARI~SOl'~' P.C. 15~053 .
2~. Pipel~ers, D.G., ar.d ~.~. Pi~eleers-Mari~hal. 1~
A method for the pur'fic~tion o~ gle A, B and ~ cells and for the isolation o couple;d cells fro~ isolate~ rat islets.
~ia~etologia 20:654-~63, 30. Pipeleers, ~.G., ~. Pipe~ee~s-Mar~'chal, J~-C. Hannaer~, M. ~ergh~a~s, P.A. Irl t t ~eld, ~. Ro~i~g, M. Van ~e Winkel, and W. ~epts. l~gl. Trans~ ta~ion of pu~ified isle~ cells in diabetic rats. I. Stand~izatio~ of islet cell gr~fts.
Diabetes 40;g0g-~lg. . : .
~1. Koxbutt, ~.S., ~d Pipeleers ~ 93. Rat panc~eas preparatlo~ for cold stor.ge ~n~ s~bsequent lslet cell isoiation. Transplantation 56:5~0-503.
32. Pipeleers D.G.. 199?. CQmpcsiti~n of islet ~r~ts.
~iabetes Nutrition an~ Meta~is~ 5 ~S~lppl. 1);29-32.
33. Rico~di C........ l~gl. ,Quant~tati~e a~d q~l~lita~i~e standards for islet ~solatior. ass~ssmen~ in hu~ans an~ large ma~r~nals. ~ancreas ~:242--24~
34. Alumets, J., R. ~akanson, ~d F. Su~dler. 1983.
Ontogeny o~ endo~ri~e ce~ls in po~cine gut Gnd pancreas. An i~muno¢ytochemical study. ~stroen~e~ology 85:1359-1372.
35. Clark, S.A., ~nd W.L. C~ ck. 1~0. Islet cell~ culture in define~ serum-free mediu~ docri~ology 1~:1895-1gO3.
36. Lir~g, Z., J.C. Hann~ert, ~nd D. Pipeleers. 1994.
~;ffect of nutrients, h~rmo~e- an:~ ser~n on survl~al ~,f .r~t i slet beta cells irl ¢ulture. Di-.be~ologi~ 37 :1S-21.
37 . RabinoYitch, A., B . B1dr~e1, T . Murray, ~d D . H . Mir~
lg80. Cyclic adenosine-3',5'-~nor~ophospha~e s~imulates islel~
B cell ~ep1ication in neorata1 ra~ parlc~e~tic ~onoi~yer cll1tures. J. Clin, In~est.. .6~;1C~5-1071.
, .
' ' ~, , -5;~

' .

OB~17~ lON 13:44 FAX ~ 78 50~ FISEI & RICEIAR~SON P.C. 1~1054 .
38. Swenne, I., A.J. Bonel S.L. Ho~ell, and C. Xeilerstr~m.
1~80. Effe~ts of glucose and amino.acids on the bios~n~hesi~
o f ~NA and insulln in fetal rat islets maihtaine~ in tissu~ ~ulture.
~ia~etes 2~:~86-~g2.
39. Hellerstr~m, C., and I.jSwenne. 1391. Fun~tional ~aturation and prol~fera~on ~f fetal p~n~reatic ~-cells.
Diabetes 40 (Su~pl. ~1:89-~3.
40. Sandler, S., and A. ~nde~s~on. lg~6. Lon~-ter~. e~e~ts of expos~re of p~nc~eatic islets to nicotinamide i~ vitro on DNA synthesis, meta~olism a~d B-c;ell function. ~iabetologia 2 g;19~-202.
41. ~chein, P.~ .A. Cooney, and M.L. Veron. 1~67. ~e ~se of nicoti~amide to ~o~y the toxi~ity of strepto~otocin d~betes w~.thout loss of an~it~mor ac~lvity. ~ancer Res ~7:2324-~332.
42. S~ndler, S., M. Welsh, and A. An~ersso~. 1983. $ t-reptozotocin-i~duced impairme~t of islet ~-~ell ~e~abolis~
and its prevention by a hy~roxyl radi~al sc~en~er a~d 2~ i~hi~itors ~f poly~A~-ri~osel synthetase. A~ta Pharmacol Toxicol (Copenhl. 53:392-400.
43. Yama~ ., K. Novaka, T. H~hafusa, T. Miyaz~ki, H.
Toyosh~a, and S. Ta~ 7. Pxeven~ive and ther~peutlc effects of large-d~se ni~otinam1de i.njecti~ns on diabetes associated wit~ in~litu~. ~ observation in nono~ese dia~etic ~NOD~ mice. ~ia~tes 3 :749-753.
44. Otcnkoski, T" ~.M. Be~tt1e, ~.I. Mally, C. Ricor~, an~
A, Hayek. 1993. Nicot-n~mide is a potent inducer of en~ocri~e dif~erentiation in cult~red human fe~al p~ncreatic cells. J. Clin. Invest. ~:1959-1466.
All referer.ces a~ p~tent do~umen~s cite~ herei~ are incorpor~ted by refere~ce as.i~ ea~h were indi~id~ally so deno~ed.

.

~. ' !
... .~ ...

0~17J~ ON 13:45 FAl~ B78 50~ FISE~ & RICHAR~SON P.C. 4Z1055 The ~oregoing description~of the invention is e~e~pl~r~
for pu~pPses of illustration ah~ expl~n~tion. It shou~d be under~too~ that vario~s modifi~atlons can be m~de without depa~ting fro~ the spirit ~nd Scope O r ~he inve~tior.
Accordingly, the ~31~ awi~g cla1ms are ~ntended to be in~er-preted to embrace all ~uch mod~ications.

-~51

Claims (42)

1. An in vitro method of isolating a substantially enriched population of cells from a tissue source comprising:
a) treating the tissue source to form a preparation of cells; and b) culturing the preparation of step a) in vitro with a serum-free basal media supplemented with a serum substitute, a agent to stimulate DNA
synthesis, and an agent to promote endocrine cell survival, resulting in a substantially enriched population of cells.

2. The method of Claim 1 wherein the cells are selected from the group consisting of islet cells, endocrine cells, neuroendocrine cells, adrenal cells, stem cells, duct cells, and hepatocytes.

3. The method of Claim 1 wherein the tissue source is selected from the group consisting of pancreas, liver, thyroid gland and blood.

4. The method of Claim 1 wherein the tissue source comprises differentiated cells and/or precursor cells.

5. The method of claim 1 wherein the tissue source is treated with an enzyme.

6, The method of Claim 5 wherein the enzyme is collagenase,

7. An in vitro method of isolating a substantially enriched population of insulin-secreting neonatal islet cells from a neonatal pancreas cells comprising:
a) treating the pancreas to form a preparation of islet cells; and b) culturing the preparation of step a) in vitro with a serum-free basal media supplemented with a serum substitute, an agent to stimulate DNA
synthesis, and an agent to promote endocrine cell survival, resulting in a substantially enriched population of insulin-secreting neonatal islet cells.

8. The method of Claim 7 wherein the pancreas is a pig pancreas or a human pancreas.

9. The method of Claim 7 wherein the serum substitute is albumin.

10. The method of Claim 7 wherein the serum substitute is albumin, the DNA-synthesis stimulating agent is isobutylmethylxanthene (IBMX) and nicotinamide, and the cell survival promoting agent is glucose and IBMX.

11. The method of Claim 10 wherein the albumin is present at a level from about 0.1% to about 1.0% weight/volume, the IBMX is present at a level from about 5 to about 100 µmol/liter, the nicotinamide is present at a level from about 0.5 to about 20 mmol/liter, and the glucose is present at a level from about 6 to about 30 mmol/liter.

12. The method of Claim 7 wherein the preparation is cultured at a temperature from about 20° to about 39°C for about 7 to about 20 days in humidified air.

13. The method of Claim 12 wherein the serum-free basal media is Ham's F10 tissue culture media, the albumin is present at about 0.5%, the IBMX is present at about 50 µmol/liter, the nicotinamide is present at about 10 m-mol/liter, and the glucose is present at about 10 mmol/liter.

14. A medium comprising a serum-free basal media supplemented with a serum substitute, an agent to stimulate DNA synthesis, and an agent to promote cell survival.

15. The medium of Claim 14 wherein the serum substrate is albumin, the DNA-synthesis stimulating agent is isobutylmethylxanthene (IBMX) and nicotinamide and the endocrine cell survival promoting agent is glucose.

16. The medium of Claim 14 which further comprises an antibiotic.

17. The medium of Claim 14 wherein the albumin is present at a level from about 0.1% to about 1.0% weight/volume, the IBMX is present at a level from about 5 to about 100 µmol/liter, the nicotinamide is present at a level from about 0.5 to about 20 mmol/liter, and the glucose is present at a level from about 6 to about 30 mmol/liter.

18. The medium of claim 17 wherein the serum-free basal, media is Ham's F10 tissue culture media, the albumin is present at about 0.5%, the IBMX is present at about 50 µmol/liter, the nicotinamide is present at about 10 m-mol/liter, and the glucose is present at about 10 mmol/liter.

19. An enriched population of endocrine cells wherein the population is produced in vitro by treating a tissue source of endocrine cells to form a preparation of cells and placing the preparation in an in vitro culture comprising serum-free basal media supplemented with a serum substitute, an agent to stimulate DNA synthesis, and an agent to promote endocrine cell survival, resulting in an enriched population of endocrine cells.

20. The population of claim 19 wherein the endocrine cells are neonatal islet cells.

21. A method of alleviating the symptoms of diabetic subject comprising administering to the subject a therapeutically-effective amount of an in vitro enriched population of insulin-secretion neonatal islet cells.

22. The method of Claim 21 wherein the neonatal islet cells are porcine or human.

23. The method of Claim 21 wherein the islet cells are microencapsulated.

24. The method of Claim 23 wherein the islet cells are transplanted into the subject.

25. The method of Claim 21 further comprising administering an immunosuppressive agent.

26. The method of Claim 21 wherein the islet cells are transfected with an exogenous gene prior to administration.

27. a method for transplantation of endocrine cells into a subject, wherein the subject would benefit from the transplantation, comprising:
a) treating a tissue source of endocrine cells to form a preparation of cells;
b) culturing the preparation of a) in vitro in a serum-free basal media supplemented with a serum substituted, an agent to stimulate DNA synthesis, resulting in an enriched population of endocrine cells; and c) transplanting the cells into the subject.

28. A method for transplantation of insulin-secreting neonatal islet cells into a diabetic subject comprising:
a) treating a pancreas to form a preparation of cells;
b) culturing the preparation in vitro is a serum-free basal media supplemented with a serum substitute, an agent to stimulate DNA synthesis, and an agent to promote endocrine cell survival, resulting in an enriched population of insulin-secreting neonatal islet cells; and c) transplanting the cells into the subject.

29. The method of claim 28 wherein the islet cells are transfected with an exogenous gene prior to transplantation.

30. The method of Claim 28 wherein the neonatal islet cells are porcine or human.

31. The method of Claim 28 further comprising administering an immunosuppressive agent pre-transplantation, concurrent with transplantation or post-transplantation.

32. The method of Claim 28 wherein the neonatal islet cells are microencapsulated.

33. An in vitro method for increasing the rate of maturation of an undifferentiated cell comprising encapsulating the cell within a stabilizing matrix or microcapsule to form an encapsulated or microencapsulated cell and culturing the encapsulated or microencapsulated cell in vitro, wherein the cell has an increased rate of maturation.

34. The method of Claim 33 wherein the microcapsule comprises alginate.

35. The method of claim 33 wherein the cell is a porcine or human neonatal islet cell.

36. The method of Claim 33 wherein the microencapsulated cell is cultured in a serum-containing or serum-free media.

37. The method of claim 33, further comprising co-encapsulating at least two cell types.

38. An in vitro method for increasing the rate of proliferation of a cell comprising encapsulating the cell within a stabilizing matrix or microcapsule to form an encapsulated or a microencapsulated cell and culturing the encapsulated or microencepsulated cell in vitro.

39. The method of Claim 38 wherein the microcapsule comprises alginate.

40. The method of Claim 38 wherein the cell is a porcine or human neonatal islet cell.

41. The method of Claim 38 wherein the microencapsulated cell is culture in a serum-containing or serum-free media.

42. The method of claim 38, further comprising co-encapsulating at least two cell types.