CA2092825A1 - Foramenous implant - Google Patents

Abstract

A chamber made of a biocompatible material, adapted to be implanted in a host, adapted to substantially contain biological material immunologically compatible with the host, having a wall, having holes traversing the wall where the holes have an inner diameter at the narrowest point large enough to permit a functional host capillary to traverse the thickness of the wall, and where said holes are numerous enough to permit said host capillary to support the viability of the contained biological material.

Description

WO 93/û263~ PCI /US92/05747 .

2~9282~

5 ~ORAMENOUS IMPLANT

FI~LD OF INVENTION
~h1s 1nvent10n re~ates to an 1mplant dev1ce and method for vascular~zat10n of 1mplanted 1mmunolog1cally co~pat1ble b101091cal materlal.
Back~round of the Invent10n Many dlseases are due to a lack or destruct10n of a spec1f1c b1010g1cal system. Examples 1nclude d1abetes ~lack of 1slet funct10n) growth retardat10n ~lack of growth hormone) other endocr1ne def1c1encles (thyrold parathyro1d reproductlve adrenal p1tu1tary) Park1nson s (lack of dopamlne) hemophllla (lack of blood clott1ng factors) and 1nborn errors of metabol1sm (m1ss1ng a spec1f1c enzyme or cofactor). In each 1nstance the d1sease state results from a lack of a spec1f1c b101091cally produced factor.
Correct10n of these d1sease states requ1res the replacement of the spec1f1c b1010g1cal funct10n. ~or example the d1abet1c must respond to m1nute to m1nute changes 1n blood glucose w1th an appropr1ate re1ease of pancreat1c hormones to ma1nta1n normal blood glucose concentrat10ns. D1abet1cs treated w1th 1nsul1n do not malnta1n normal blood glucose levels. Implantat10n of funct10nal 1slets of Langerhans eures the d1sease 1nsul1n therapy merely treats the dlsease.
Se~veral approaches to 1mplant1ng 11v1ng cells or t1ssue and dev1ces have been attempted. Vacant1 and Langer have developed a system descr1bed 1n ~Ch1mer1c Neomorphogenesls of Organs by Controlled Cellular Implantat10n Us1ng Art1f1c1al Matr1cesn WO
Patent Appllcat10n No. 8B/03705. ~h1s system 1s a tree-11ke matr1x of b10degradable polymer on wh1ch cells are grown for , , . ' ~ . . ' . , , ~ . : .
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WO 93/~263~ PCT/US92/0~7 17 , ~ o 9 ~ 8 ~ 5 2 subsequent 1mplantat10n. The dev1ce 1s 1mplanted 1nto ,the pat1ent, host vascularlz3tlon of the cells occurs, the matrSx ~s desraded and a psuedo-organ theoret~cally rema1ns. The formatlon of the f1nal funct~onal organ requ1res a complex 1nteractlon between the dev1ce and host 1n an undeflned and uncontrollable manner. Th1s dev1ce, should 1t fall for any reason, ~ould be d1ff1cult to remove due to the extens1ve connectlons to the host.
Another approach for the format10n of organ-11ke structures 1n 1mmunolog1cally compat1ble pat1ents 1s descr1bed by Thompson, lO Anderson, and ~ac1ag 1n ^Dev1ce for D1rected Neovascular1zat10n and Method for Same,~ W0 Patent Appl1cat10n No. 89/07944. The key features of th1s approach are a b10compat1ble matr1x, s1mllar 1n structure to that of Vacant1 and Langer, a coat1ng of ang10genlc growth factors whlch are used to 1nduce 1n vlvo d1rected 15 neovascular1zat10n and subsequent 1mplantat10n 1n pat1ents. The result1ng structure, termed an organo1d, may be useful as an 1mplantat10n slte for cells of therapeut1c 1mportance.
Alternat1vely, 1t may be poss1ble to attach cells pr~or to ~mplantat10n.
Valent1n1 et al., U.S. Patcnt No. 4,877,029 descrlDes a sem1permeable nerve gu1dance channel ~h1ch 1s composed of a channel w1th a cell 1mpermeable smooth 1nner membrane face and an outer surface ~1th long1tudlnally d1rected trabeculae 1n the s1ze range of l to 20 m1crons. Thls trabecular conf1gurat10n does not 25 1nclude holes ~h1ch traverse the th1ckness of the dev1ce and does not allo~ vascular growth 1nto the 1nner compartment.
Ronel et al., U.S. Patent No. ~,298,002 descr1bes a porous hydrophll1c chamber for 1mplant1ng cells. Thls devlte does not permlt the 1nvas10n of vascular elements ~1th1n the chamber.
Dental and bone 1mplants ~lth pores ~n the S to 50 mlcrons s1ze range allo~ f1brotlc 1ngro~th to anchor the 1mplant ~1th~n t1ssue, but do not prov1de a chamber for 1mplantlng cells and do not result 1n vascular1zat10n of 1mplanted cells or t1ssue.

W O 93t02~3~ PCT/~IS92/05747 , .
20~2825 Add~t10nal examples of pr10r.art 1nclude U.S. Patent No.

4,553,272 ~Regenerat10n of L1v1ng ~1ssues by Gro~th of Isolated Cells 1n Porous Implant and Product Thereof" and U.S. Patent No.
4,378,016 Art1f1c1al Endocr1ne Gland Conta1n1ng Hormone Produc1ng S Cells.~

SUMMARY 0~ ~HE INVENTION
Appl1cants 1nvent10n 1s a chamber made of a blocompatlble mater1al adapted to be 1mplanted 1n a host, and to substant1ally ; 10 conta1n b1010g1cal mater1al tmmunologtcally compat1~1e ~1th the host, sa1d chamber havlng a ~all, sa1d ~all hav1ng holes travers1ng the ~all where the holes have an 1nner d1ameter at the narrowest po1nt large enough to permlt a host cap111ary to traverse the thlckness of the wall, and ~here sald holes are lS numerous enough to perm1t sa1d host cap111ary to support the ! v1ab~11ty of the b1010g1cal mater1al whlch may be conta1ned there1n.
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BRIE~ DESCRIPTION 0~ rHE ORAWINGS
~1gure l. Cross sect10n dra~1ng of a chamber used for vascular1z1ng t1ssue ~1th1n the ehamber. The chamber 1s made of an outer hous1n~, a foramenous membrane, o-r1ng spacer and seal1ng r1ng.
F19ure 2A. Bod~ ~e1ght changes of rats 1mplanted ~1th large hole chambers ~70 x 110 mlcron holes). Ind1vtdual body ~e1ght changes of 3 rats 1mplanted ~1th large hole chambers ( ~ , ~ , ~ ) are compared to sham controls ( O , N.3, mean ~ SEM) and dlabet1c controls ~ ~ , N.3, mean ~ SEM). One d1abet1c an1mal d1ed at 3 ~eeks. Yalues after 3 ~eeks are the mean of the 2 rema1n1ng d1abet1c controls. Each rat ~as 1mplanted on day O ~lth 2 chambers each conta1n1ng 2000 1slets. ~embranes were punctured ~1th a 30 gauge needle ~70 x 110 m1cron holes) to form the large holes. All chambers ~ere 1mplanted tnto ep1d1dymal fat pads.
Chambers were removed as 1nd1cated by ~

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W O 93~0263~ PC~r/US92/05747 209'~2~ 4 _ Elgure 2B. Body we1ght changes of rats 1mplanted ~1th 1ntact (3 m1cron pore) ch~mbers. Ind1v1dual body we1~ht changes of 3 rats lmplanted w1th 1ntact membranes compared to the sham ( 0 ~N=3) and dlabet1c controls ( ~ H=3). An1mals were 1mplanted exactly as 1n F1gure 2A except the chdmber conta1ned 3 mlcron pores.
~ lgure 3. Hon-fast1ng blood glucose concentrat10ns of rats 1mplanted w1th large hole chambers (70 x llO m1cron holes). Blood glucose concentrat10ns of the 3 an1mals w1th large hole dev1ce 1mplants are compared to sham ( ~ N=3) and alabet~c control ( O N-3) an1ma1s.
F1gure 4. Non-fast1ng blood glucose concentrat10ns of rats 1mplanted w1th 1ntact (3 m1cron pore) chambers. Blood glucose concentrat10ns of the 3 an1mals 1mplanted w1th 3 mlcron pore membrane are compared to sham ~ O N~3) and d1abet1c control N=3) an~mals.
Flsure 5. Intraper1toneal glucose tolerance test (IP GTT) of rats 1mplanted wlth large hole thambers (70 x llO mlcron holes).
Results are the mean ~ SEM of the curves for the 3 anlmals 1mplanted w1th large hole chamDers before 1mplant ( ~ eek before explant ~ o ) and l week aftcr explant ~
~ 19ure 6. Intraper1toneal glucose tolerance test ~IP GTT) of rats 1mplantad w1th 1ntact (3 m1cron pore) membranes. The results a.re the mean of the IP 6T~ curv-s of the 3 an1mals before 1mplant ( ) l week before explant ( O ). and l week after explant ( ~ ).
~1gure 7. Hematoxyl1n/eos1n sta1ned sect10ns of a large hole ; chamber (70 x 110 mlcron holes). Thls chamber was explanted after 4 weeks. Numerous large 1slet masses were ev1dent 1n th1s large hole chamber. Extenslve vasculature had 1nvaded the t1ssue w~th c~p111ar1es ev1dent ~1thln 1slets themselves. S1mllar results were noted from dev1ces exDlanted ~t 7 and 9 wee~s.

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WO 93/0263~ PCr/US92/0~741 -- 5 - 209282~

Flgure 8~ Hematoxyl1n/eos1n sta~ned sectlon of an lntact (3 m~cron pore) membrane chamber. Th1s chamber ~as explanted after 4 weeks. Desplte the extens1ve vascular response surround1ng the membrane only a few small 1slet areas were present w1th1n the chamber demonstrat~ng the ~nab~11ty of the 1ntact (3 m~cron pore) chamber to support the tslet.
Flgure 9 L19ht mlcrographs of 14 day fetal syngene~c lung t1ssue after 1mplantat10n for 21 days 1n a chamber ~1th 5 mlcron pores. Note the ~ell dlfferent~ated h1ghly vascularlzed tlssue ~1th1n the chamber. Th1s pore s1ze prevented the outgro~th of the t1ssue ~1th1n the chamber.
F1gure lO. Llght mlcrographs of 14 day fetal syngene1c lung t1ssue after 1mplantat10n for 21 days 1n a chamber ~1th lO to 15 mlcron pores Note the well d1fferent1dted~ h19hly vascularlzed lS t1ssue w1th1n the devlce. Th1s chamber also prevented the outgrowth of the 1mplanted t1ssue.
f1gure ll. L1ght m1crographs of recomb1nant t1ssue after 1mplantat10n 1n ep1d1dymal fat pads for e days ln a chamber w1th large holes ~9û x 170 mlcron holes). The recomb1nant sho~s pos1t1ve 1mmunocytochem1cal sta1nlng for 1nsul1n ~A) and glucagon (B). In addlt10n there 1s prol1f1c morphogenes1s of tubules and a h1gh degree of vascularlty.
F1gure t2. L1ght m1crographs of pancreatlc rud1ments after 1mplantat10n 1n ep1d1dymal fat pads for 8 days 1n a chamber w1th large holes ~90 x 170 m1cron holes). Pancreat1c rud1ments sho~
1nsul1n ~A) and glucagon (B) pos1t1ve cells tubular morphogenes1s and a h19h degree of vascular1ty.
F1gure 13. H1stolog1cal appearance of another large hole chamber (70 x llO m1cron holes) after 3 days ~mplantat~on.
Chambers conta1n1ng about 500 1slets 1n Lew1s rats produced a rap1d vascular response as sho~n by the large vessel cours1ng through the center of the chamber after only 3 days 1n the an1mals.

.- - , w o 93/0263~ PCT/~S92/057~

~9282~ 6 -f1gure 14. H~stolos1cal appearance of ~ m~cron pore chamber after 3 days 1mplantat10n. Dev1ces conta1n1ng about 500 1slets 1n Lew1s rats lacked an 1nttmate vascular1zat10n response w1th1n the t1ssue chamber.

DETAILED DESCRIPTION OF INVENTION
Th1s 1nvent10n 1s a thamber for substant1ally conta1nlng tells, cellular organelles or t1ssue. The chamber 1s made of b10compat1ble mater1al adapted for 1mplantat10n 1n a host. The chamber ts constructed ~1th holes that traverse the ent1re th1ckness of the chamber wall. The holes need not be stra1ght or un1form. They may form an 1rrgeular pathway from the exter10r to the 1nter10r of the chamber. The holes have an 1nner d1ameter large enough to allow the 1ngrowth and egress of host cap111ar1es lS to and from cells or t1ssue 1n the chamber. Th1s dev1ce 1s termed here1n a foramenous chamber. Th1s chamber allows the host vasculàr system to support both v1abll1ty and therapeut1c effect1veness of b1010g1cal materldl placed 1n the chamber. The cnt1re chamber need not have holes 1n 1t so long as d port10n of the chamber has enough holes to support the v1ablllty of the conta1ned mater1al. The upper 11m1t of the hole s1ze w111 vary w1th the part1cular appl1cat10n. If the b1010g1cal mater1al 1s to be completely conta1ned 1n the chamber then the upper 11m1t of the hole s1ze w111 be determ1ned by the s1ze of the cells or mater1al conta1ned 1n the chamber. If complete conta1nment 1s not necessary, then the holes may be any s1ze wh1ch w111 substant1ally conta1n the b1oloslcdl mater1al. If the mater1al also contalns some holes smaller than that necessary to allow cap111ar1es through, 1t w111 not 1nterfere w1th the operat10n of the chamber.
Such smaller holes need not be avo1ded. The conta1ned b101091cal mater1al may be any cell, cell comb1nat10n, organo1d, organ or t1ssue to be 1mplanted 1nto a host. The b1010g1cal mater1al 1s preferably 1mmunolog1cally compat1ble w1th the host. The chamber , '' ~ -.
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w u ~/u ~ PCT/US92/0~7~7 .
_ 7 _ 209282~

may be any host blocompat1b7e mater1al thdt can be safely ~mplanted 1nto the host. See for example Tab7e I, page x1, 1n Conc1se Gu1de to B~omed1cal Polvmers ~y J. w. Boretos ~.9-73), 11stlng stable and sem1stable mater1als appropr1ate for use 1n S th1s 1nvent10n.
In the past, other attempts to lmplant tlssùe w1thln a devlce 1nto a host whlle tak1ng advantage of the host vasculature have shown poor cell v1ab111ty because nutr1ents were not made ava11able to the cells at a suff1c1ent rate. See Scharp, D. ~. et al., ~Islet l~muno-lsolat10n:The Use of Hybr~d Art1flcal Organs to Prevent Islet T1ssue Re~ect~on" 1n World Journal of Sùrgery, Volume ~, pp. 221 to 229 (1984). In accordance w1th the present 1nventlon the slze and number of holes are sufflc1ent to support early and sustalned cell v1ab111ty becausé enouqh cap11iar1es form lS rap1dly enough follo~1ng ~mplantat10n tc. allo.~ the prov1s10n of nutrlents at an appropr~ate rate.
The eha~ber may be used for tlssue or cell replacement ln the correct10n of dlsease states. Allograft 1mplants of cellular organelles or free cells to correct a dlsease state are currently pcrformed most commonly by lnfus10n lnto the portal c1rculatlon to allow the tells to lodge 1n the 11ver. The 1nstant 1nventlon allows use of an alternate 1mplant s1te that can be easlly atcessed and the chamber w1th 1ts contents may be removed 1f n~cessary. The chamber allows the host to prov1de adequate nutr1t1nal support for the 1mplanted t1ssue to correct a d1sease state. Accord1ngly, the chamber may be used for allograft transplant of human t1ssue.
It may also be used to 1mplant a pat1ent s own cells whkh have been genet1cally altered so that they produee a therapeut1c product. Once cells have been transformed to express a gene and secrete a therapeutlc product, a removable contalner for those cells 1s des1rable.

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WO 93/0263; PCr/US92/0~7 1 2 0 9 ~ 8 2 5 _ 8 -Th1s chamber may also be used to contruct a hybr1d bloart1f1c1al organ. The chamber allows the host to supply the bloart1flc1al organ w1th an 1nt1mate vasculature so that nutr1ents are del1vered to the organ, metdbol1c wastes are removed, and the therapeut1t product made by the organ 1s del1vered to the host.
The chamber can be used to enclose an lnner 1ntact cellular protectant membrane. Vascular e1ements penetrate the chamber and provlde suff1c1ent nutr1ent and waste exchange for the enclosed cells to surv1ve and funct10n to correct a d1abet1c an1mal.
The chamber can also be uset to conta1n m'croencapsuldted cells or organelles to be 1mplanted. By tmplant1n~ the encapsulated t1ssue w1th1n the thamber a rap1d vascular1zat10n w111 be st1mulated and the capsules w11,1 be conta1net w1th1n a def1ned space for easy removal should the need ar1se.
lS In one embod1ment of the 1nvent10n the follow1ng components were used: a foramenous membrane; a hous1ng to hold the membrane;
an o-r1ng to prov1de d t1ssue space; and b1010g1cal t1ssue (~1gure l). The structural hous1ng supports the membrane and provldes 1ntegr1ty. In a prefered embod1ment 1t 1s made of t~tan~um.
However, hous1ngs of Teflon, polystyrene and polypropylene have also been made and the hous1ng can be made from atternat1ve b10compat1ble mater1als. rhe o-r1ng of appropr1ate th1ckness, such as about lO to 250 m1cron, prov1des a space for cells or t1ssue. ~yp1cally the o-r1ng 1s made of s111cone although o-r1ngs of other b10compat1ble mater1als may be used.
~embranes w1th a nom1nal pore s1ze of about 3 m1cron fa11ed to 1nduce a rap1d vascular response w1th1n the rhamber (~1gure 14).
Vascular elements were detected 1n these 3 m1cron pore membrane dev1ces after 3 to 9 weeks of 1mplantat10n, but the vessels were occluded w1th 1nadequate blood flow to the 1mplanted t1ssue. Th1s WdS shown by 1slets 1mplanted w1thln ~ 3 m1cron pore membrane ?.

WO 93/0263~ PCI`/US92/057~ /
-, - 9 - 2~92825 dev1ce fa111ng to correct d1abet1c-anlmals ~F1gures 2b, 4, 6, and B). Thus, the lower 11mlt of nom1nal pore s1ze to lnduce an adequate vascular response ts greater than about 3 m1cron.
Membranes w1th nom1nal pore s1ze of S to 15 m1cron 1nduced a rap1d S vascular response (~1gures 9 and lO). Holes up to and 1nclud1ng those of 90 x 170 mlcron 1nduted the response (F19ures 7, ll, 12, and 13).
Alternat1ve forms of the chamber are also poss~ble. These 1nclude, but are not 11m1ted to, 1nclud1ng a scaffold1ng for cellular attachment w1th1n the chamber; prov1d1ng an 1ntact 1mmunoprotect1ve membrane enclos1ng cellular elements w1th1n the chamber; us1ng m1croencapsulated t1ssue ~1th1n the dev1ce; mak1ng the o-r1ng of a mater1al wh1ch w111 seal the membranes together el1m1nat1ng the need for the hous1ng; stacked membrane packets to prov1de add1t10nal space for 1mplanted t1ssue; mak1ng the dev1ce 1n the form of hollow f1bers; or seal1ng the membrane w1th techn1ques such as son1c weldlng that does not requ1re a hous1ng.

~XAMPLE I
In order to mon1tor the ab111ty of the chambers to correct d1abetes, d1abet1c Lew1s rats were 1mplanted w1th large hole membrane or 1ntact membrane dev1ces conta1nlng 1solated syngene1c 1slets. Implantet anlmals were compared to sham operated and dlabet1c control an1mals.
Membrane PreDarat'on and Dev1ce Descr1Dt10n D1scs of the test membrane la teflon membrane wlth polyester f1ber back~ng, 3.0 m1cron effectlve nom1nal pore s1ze, Gore 3 m~cron teflon ~Ll0956) were sequent~ally washed ~n ~reon-TE35 for 30 to 60 mlnutes, several changes of ethanol and then soaked for 45 m1nutes 1n ethanol. The ethanol was removed w1th a ster11e sal1ne wash 10.9% NaCl). Membr3nes were transferred to culture med1um (RPMI 1640 ~ lOX ~etal Bov1ne serum, ~BS) and placed 1n a ,.~ ....... .. . .

: - ;,. -, .
- , w o 93/0263~ PCT/~IS92/0~7~-20~282~ - 10 -37C C02 1ncubator. Half of the membranes ~ere le~t tntact (3 mlcron pores) and half were perforated 24 t1mes w1th a 3~ ster11e needle ~large hole membranes, pore stze 70 x 71~ mlcron). Devtces for holdlng the 1slets cons1sted of a tttan1um chamber ~th S fr1ct10n closure f1tted w1th 2 test membrane d1scs separated by a 120 mlcron thlck o-r1ng (E1gure l). T1tan1um chambers and sll1con o-r1ngs were ster111zed by soak1ng tn ethanol and r1nsed 1n ster11e sal1ne be~ore use.

Induc1nq Dtabetes D1abetes was lnduced by 90X pancreatectomy ~Px) follo~ed by I.V. 1nJect10n w1th streptozotoc1n ~Stz) at 60 mg/kg body ~elght v1d the ta11 ve1n 3 to lO days post-pancreatectomy.

Adult Rat Islet Isolat10n Islets from syngenelc Lew1s rats were used for 1mplantat10n.
Eollow1ng sacr1f1ce, under dsept1c cond1t10ns, the common b11e duct was cannulated and the pancreas 1nflated w1th 10 ml of Hank s balanced salt solut10n (HBSS). The 1nflated pancreas was removed, cleaned dnd m1nced. ~lnced t1ssùe was d19ested ~1th collagenase ~type V, 51gma) at lO mg per pancreas 1n a 39-C water bath.
D1gest10n was stopped w1th lce-cold HBSS and the contents ~ashed 3 t1mes w1th HBSS, Islets ~ere separated from ac1nar and most ductal ttssue by centrlfugat10n 1n a bov1ne serum album1n (BSA) step grad1ent of 29%, 26%, 23%, and 20%. Islets ~ere ~ashed ~1th RPMI
1640 med1um w1th 10% fetal bov1ne serum and cultured at 37-C 1n a1r/5% C02 for 3 to 7 days before 1mplantatton.

Dev1ce ImDlanatton Islets were ~and p1cked from the culture plates for load1ng the dev1ces. Ten ul of 1slet suspens10n ~approx1mately 2000 1slets) were loaded 1nto each test devlce. Each anlmal rece~ved 2 devtces ~4000 lslets total). Antmals were anesthet1zed w1th .. , ................ ~ : ............ : .: . --.
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xylazlne (S mg/kg) and ketam1ne (65 mg/kg) and the1r eptd1dym21 fat pads 1soldted through a small lower abdom1nal 1ncls10n. One dev1ce was placed on each fat pad and covered w1th the fat pad.
Dev1ces were held 1n place wlth Nexaband (CRX ~ed1cal, Inc.) t1ssue adhes1ve. The abdomlnal muscle was closed wlth suture (4-0 gut) and the sk1n w1th sta1nless steel wound c11ps.

Assessment of D1abetes Correct10n The funct10nal state of the anlmals was evaluated by mon1tor1ng the follow1ng parameters: change 1n body we19ht, non-fast1ng blood glucose, 1ntraper1toneal glucose tolerance, and h1stology of the 1mplants and pancreatlc remnants at the t1me of sacr1f1ce. Per10d1c non-fast1ng blood glucose levels ~ere assessed w1th an ExacTech~ Blood Glucose ~eter (Med1sense) 1n blood obta1ned from a ta11 ve1n st1ck. The upper 11mlt of detect10n of the ExacTech~ Blood 61ucose Meter 1s 450 mg/dl.
Values reported as 450 mg/dl are a m1nlmal est1mate of the actual blood glucose concentratlon. Intraper1toneal glucose tolerance tests (IP GTT) cons1st of an overn1ght fast, I.P. 1n~ect10n of glucose (2 g/kg body we1ght from a 20X glucose solutlon) and measur1ng blood glucose at O ~before 1nJectlon), lO, 30, SO, and 90 m1nutes. Body we19hts were mon1tored throughout the study.

. Dev1ce Removal Dev1ees were explanted after ~, 7, and 9 weeks of 1mplantat10n. Explantat10n cons1sted of 1solat1ng the ep1d1dymal fat pads, 11gat1ng ~1th s11k suture and remov1ng the dev1ces.
Dev1ces were f1xed for h1stology, one 1n glutaraldehyde the other 1n Bou1n s f1xat1ve. Eollow1ng explant, non-fast1ng blood glucose levels, fastlng IP 6TT s and body we1ghts were mon1tored to assess revers~on to the d1abet1c state 1n those an1mals that had shown correct10n. After reversion was establ1shed, the an1mals were sacr1f1ced, the pancreat1c remnant removed and f1xed 1n Bou1n s for h1stolog1cal examlnat10n.

WO 93/0263~ PCT/US92/05747 . .
209282~ 12 -Four groups ~N=3 per group) of anlmals were mon1tored: sham operated controls, dlabet1c controls, large hole membrane chamber lmplants (70 x 110 m1cron holes), and 1ntact membrane chamber 1mplants ~3 m1cron pore).
s Bodv Wetqht Chanqes All 3 an1mals 1mplanted ~1th large hole membrane chambers ga1ned ~e1~ht at a rate comparable to or greater than sham controls (~1gure 2A) dur1ng the per10d of 1mplantatlon. In contrast, only one of the 3 an1mals 1mplanted ~1th 1ntact membrane chambers galned welght comparable to control rats (F1gure 2B).
The d1abet1c control an1mals fa11ed to ga1n we19ht.

Non-Fast1nq ~lood Glucose lS Large hole chamber 1mplants reduced non-fast1ng blood glucose levels 1n all three an1mals to control levels (Flgure 3). In all 3 anSmals, blood glucose levels rapldly rose to d1abet1c levels after dev1ce explantat10n. An1mals 1mplanted w1th 1ntact membrane chambers had only sl1ght, errat1c reduct10ns 1n non-fast1ng blood glucose after 1mplantat10n ~F1gure 4). In no 1nstance was glucose reduced to sham values. In fact, blood glucose rema1ned at d1abet1c values.

. IntraDer1toneal Glucose Tolerance Tests tI~ 3U
Islets 1n the large hole 1mplants ~ere funct10nally 1ntact as shown by the1r acute response to a glucose challenge (F1gure 5).
Wlth the large hole 1mplants 1n the an1mals, the1r I~ GTT curve was 1dent1cal to the sham control curve. However, before lmplant ant after explant the1r IP GTT curve was 1nd1st1ngu1shable from the d1abet1c curve. In contrast, the 1ntact membrane 1mplants aemonstrated only 11c1te~ funct10n ~F1gure 6).

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WO 93~0263~ PCI/US9Z/0~7~, - 13 _ 2~9282~ -H1 stol ~qv The large hole chambers conta1ned large numbers of well vascularlzed 1slets ~F1~ure 7). Cap111arles were dete ted ~1th1n the 1slets themselves. The 1ntact membrane 1mplant was also well S vascular12ed on both the lns~de and outslde of the chamber, but large blood 1slands formed w1th1n the dev1ce 1ndlcat1ng a lack of freely flowlng blood. In add1t1nn, only a small number of 1slets were found wlth1n the chambers (~19ure ~). Res1dual pancreas w1thln the dlabet1c an1mals only contalned sparse, badly damaged 1slet remnants.
The body we1ght changes, non-fast1ng blood glucose levels, IP
GTT s, and h1stology demonstrate that the large hole 1mplants corrected the d1abetes of these 3 an1mals. The same parameters 1ndlcate that the 1ntact membrane 1mplants had only 11m1ted funct10n and d1d not correct the d1abet1c state of the 3 an1mals w1th 1ntact 1mplants. The 1slets were malntalned 1n a functlonal state 1n the large hole chambers because the chimbers allow vascular1zat10n of the 1slets through the large holes 1n the chamber.

Implantat10n of fetal lung t1ssue 1n Gore- S m1cron teflon ~#X12039) membranes and Gore- lO to 15 m1tron teflon (#Xl2299) mçmbranes w~th polyester back~ng.
Lung t1ssue from day l~ of gestat10n was 1solated, m1nced 1nto approx1mately l mm3 p1eces, ~ashed w1th DMEM, 20X FBS and placed 1n t1tan1um chambers. The membranes used for th1s exper1ment were teflon w1th nom1nal pore s1ze of e1ther S or lO to 15 m1cron.
Dev1ces were placed 1nto ep1d1dymal fat pads, left 1n the anlmals 3û for 21 days and then explanted. T1ssue was f1xed 1n 2.5%
glutaraldehyde and processed for h1stolog1cal exam1ntt10n.
Membranes w1th 5 m1cron nom1nal pore s1ze allowed the fetal lung t1ssue to t1fferent1ate and mature and s1gn1f~cant vascular .
.

' , .' W o 93io263~ PCT/~S92/0574/

209282~ 74 resp~nse to the conta~ned t1ssue was evldent (F1gure ~). Note that w1th the 1ntact 5 m1cron pore membrdne the t1ssue was conta1ned w1th1n the dev1ce and dtd not grDw out of the pores. -Membranes w1th ~0 to 15 m~cron nomlnal pores also perm1tted the full d1fferent1at10n and mdturat1on of the 1mpl3nted t1ssue ~F1gure 10). Aga1n t1ssue was conta1ned w1th1n the 1nter10r of the chamber and ~as well vascular1zed.

~mplantatlon of fetal pancreat1c buds and tlssue recomblnants of fetal mesenthymal dnd ddult ductal eplthellum.

Gastr1c mesenchymal t1ssue and pancreat1c rud1ments were 1solated from day 14 gestat10n Le~1s rats and pancreat1c ductal ep1thel1um was 1so1ated from adult Lew1s rats. Recomb1nants were formed by carefully p1pett1ng 1solated ductal ep1thellum onto p1eces of fetal mesenchyme ~1th1n the t1ssue chamber dev1ce.
Membranes used for these exper1ments were 0.45 m1cron pore teflon (M1111pore CM) plerced w1th a 26 gauge needle 24 t1mes to produce large holes (90 x 1~0 mlcron) 1n the upper membrane. The lower membrane ~as left 1ntact.
The tlssue chamber dev1ces ~ere lmp anted on the ep1d1dymal fat pad of adult male Lew1s rats. The epld1dymal fat pad ~as pulled through a med1al 1nc1s10n Just anter10r to the pen1s and la1d on ster11e gau~e ~etted ~1th sal1ne. The chamber ~as placed on the fat pad and the t1tan1um r1ng ~as glued to the fat pad w~th t1ssue adhes1ve.
Recomb1nants (N-10) and pancreat1c rud1ments (N.6) were ~mplanted for a days or 6 to 12 weeks. Chambers ~ere explanted from the Lewls rats and f1xed 1n eoutn s. After process1ng sect10ns . ~ere sta1ned ~1th hematoxyl1n and eos1n and 1mmunocytochem1cally sta1ned for 1nsul1n and glucagon ~here appropr1ate.

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wo 93/0263~ PCl /I,'S92/0~747 .
~s 20~282~ i , ~ .
Recomblnant t1ssues demonstrated stgn1f~cant t1ssue development as evldenced by the format10n of tubules s1milar to those of 1ntact pancreas. Cells 1mmunoposlt1ve for 1nsul1n and glucagon were detected after 8 days of 1mplantat10n (F1gure ll), S demonstrat1ng that d1fferentlatlon had occurred. Slm11ar morphogenesls was ev1dent 1n the t1ssue explanted af~er 6 to 12 weeks. Endocr1ne t1ssue also d1fFerent1ated 1n pancreat1c rud1ments as ev1denced by 1mmunostaln1ng for 1nsul1n and glucagon after B days 1mplantat10n ~F19ure 12). In all 1nstances the 1mplanted t1ssue ~as well vascular~zed. Thus, the large hole membrane chambers were capable of support1ng fetal t1ssue growth, morphogenes1s and d1fferentlat10n due to the substant1al host vascular1zat10n.

Rap1d vascular1zat10n of 1solated 1slets 1n large hole chambers (70 x llO mlcron holes).
Membranes used for th1s exper1ment ~ere 3 mlcron nom1nal pore teflon membr~nes (Gore- 3 mlcron Teflon #Ll0956) left 1ntact ~3 mlcron) or p1erced w1th 70 x llO m1cron holes. Approx1mately 500 pancreat1c 1slets 1solated from normal adult Lew1s rats ~ere placed w1th1n chambers 1n each conf1gurat10n ~N-2 per group). The chambers were 1mplanted 1nto the ep1d1dymal fat pad of normal Lew1s rats, explanted and exam1ned h1stolog1cally 3 days later.
At day 3 post-1mplantat10n wlth the large hole membrane chamber vessels ~ere detected cours1ng through the 1nter10r of the chamber around the 1mplanted 1slets (F1sure 13). In contrast, 1slets 1mplanted w1th1n the 3 m1cron pore chamber fa11ed to be vascular1zed at 3 days post-1mplant ~F1gure 14).
~0 It should be understood that var10us changes and mod1f1cat10ns to the presently preferred embod1ments descr1bed here1n w111 be apparent to those sk111ed 1n the art. Such changes and ~ :.

~ ~ 7J~ U 'Uj~ PCT/US92/05747 2~9~8~5 16 -modlf1cattons can be made ~tthout departtng from the sp1r1t and scope of the present 1nvent10n and ~1thout d1m1n1sh1ng 1ts attendant advantages. It 1s therefore 1ntended that such changes and modlf1cat10ns be covered by the appended cla1ms.

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Claims (16)

1. A chamber made of a biocompatible material (a) adapted to be implanted in a host, (b) adapted to substantially contain biological material immunologically compatible with the host, (c) having a wall, (d) having holes traversing the wall where the holes have an inner diameter at the narrowest point large enough to permit a functional host capillary to traverse the thickness of the wall, and (e) where said holes are numerous enough to permit said host capillary to support the viability of contained biological material.

2. The chamber of Claim 1 where the inner diameter at the narrowest point is greater than about three microns.

3. The chamber of Claim 1 where the inner diameter at the narrowest point is small enough so that the biological material is substantially contained in the chamber when implanted in the host.

4. The chamber of Claim 1 where the biocompatible material is teflon.

5. The chamber of Claim 1 in the configuration of a hollow fiber.

6. The chamber of Claim 1 containing living cells.

7. The chamber of Claim 6 where the living cells are capable of secreting a therapeutic product.

8. The chamber of Claim 6 where the living cells are pituitary cells, pancreatic islet cells, pancreatic beta cells, thyroid cells, parathyroid cells, or adrenal medullary cells.

9. The chamber of Claim 7 where the living cells are growth hormone secreting cells, Factor VIII secreting cells or Factor IX
secreting cells.

10. The chamber of Claim 1 having input and output ports for access to the contents of the chamber.

11. The chamber of Claim 6 where the living cells are microencapsulated.

12. The chamber of Claim 1 containing a matrix on which cells may adhere and grow.

13. The chamber of Claim 1 where the biological material immunologically compatible with the host is allograft or xenograph tissue contained in an immunoisolating membrane.

14. A method comprising implanting the chamber of Claim 6 into a host.

15. A method comprising implanting the chamber of Claim 7 into a host.

16. A method comprising implanting the chamber of Claim 13 into a host.