IN VITRO AND IN VIVO ASSESSMENT OF MICROENCAPSULATED pTC6-F7 CELLS

Salim Mamujee

A thesis subrnitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Physiology

University of Toronto

O Copyright by Salim Mamujee 1997

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In Vitro and In Vivo Assessrnent of Microencapsulated pTC6-F7 Cells

Master of Science. 1997 Salim Mamujee

Graduate Department of Physiology, University of Toronto

Abstract In order to serve as a mode1 for an engineered insulin-secreting cell. BTC6-F7

cells. derived fiom insulinomas arising in transgenic mice. were encapsulated within a

semi-permeable. alginate-poly-L-lysine alginate (APA) membrane. In this study, the in

vitro growth and insulin secretion fiom microencapsulated PTC6-F7 cells were

determined. In vitro. the insulin response to glucose. arginine. isobutylrnethylxanthine

(IBMX) and glucagon-like peptide (7-36) were also measured. In addition,

microencapsulated PTC6-F7 cells were intraperitoneally implanted into streptozotocin

diabetic rats to assess their function in vivo.

Results indicate that the encapsulation process exerts no deleterious effects on ce11

viability and the APA microcapsule provides an environment compatible with ce11

proliferation and insulin secretion. Furthemore. microencapsulated PTC6-F7 cells have

demonstrated their efficacy in reversing diabetic hyperglycemia and restoring a rapid

glucose clearance in diabetic grafi recipients. This study shows the potential use of a

eenetically engineered insulin-secreting ce11 protected within an APA microcapsule to - provide endogenous insulin therapy is feasible.

Ta mj. dearesr puï-ents. .\'oordi and Furidcrr und mj. sis fer Shubnurn:

for a liferime of sacrifice. rupporr und unconditionul love.

Acknowledaements I wish to extend my mon sincere gratitude to Dr. Sun for giving me the

opportunity ro work in his lab. His guidance. supervision. and encouragement have

proven invaluable.

Many th& to Dr. Daobiao Zhou for the endless hours of patient teaching and

demonstrating various techniques and always k i n g uilling to demonstrate yet again !

1 also wish to express my gratitude to Dr. Ivan Vacek for al1 the time spent in

reading. editing and providing suggestions. His cheemilness and eagemess to assist wiil

always be remembered.

A final word of thanks to my supervisop cornmittee: Dr. .Michael Wheeler and

Dr. Hon Kwan for their advice. comments and suggestions.

Table of Contents

List of

List of

Abbreviations ....................................

Figures ...........................................

I . Introduction 1 Diabetes: Etiology .............................................................................

I . Pathogenesis of T-vpe I Diabetes ......................................... Insulin Secretion and P-cell function .................................................

1 . Insulin gene regulation ........................................................ 2 . Insulin biosynthesis ............................................................. 3 . Nutrient induced insulin secretion ................................

Diabetes: Therapy ............................................................. Alternative strategies for insulin replacement in T > Q ~ I Diabetes .......

I . Whole pancreas transplants .................................................. 2 . Isolated islet transplantation .................................................

Protection from the host's immune system ...................................... I . Immunomodulation ............................................................... 2 . [mmunosuppression ............ ... ........................................... 3 . tmmunoisolation ........................................................

Xenotransplantation and Encapsulation .............................................. I . Microencapsulation ......................................................... 2 . Macroencapsulation ..... ..., .....................................................

Cell therapy .............. ... ................................................................. Insulin secreting cell lines .................. ... .........................................

1 . Induced insulinomas in vivo ................... .... .................... 2 . Transformation of islets in vitro .......................................... 3 . Insulinomas derived From transgenic animals .......................

Insulinorna cell immobilization ...........................................................

I I I . Experimental Design & Methodologv 16 A . Materials and Methods ........................................................................

1 . Cell culture .......................... .. ............................................. 2 . Cell encapsulation ...................................

............................................................................. . 3 Cellplating 4 . ChaIlenge studies .......................... ,.., .................................... . ...................................*..*...*.**................*-*.............. 5 Cell count

B . fn virro encapsulated PTC6-F7 cell response to secretagogue ............. 1 . /n vitro experiment methodology ........................................... 2 . In virro growth and secretion ................................................. 3 Response to Arginine ........................................................ .... 4 . Response to sub-physiotogica1 levels of glucose ................... - 3 . Response to glucose over 2 1 days ......................................... 6 . Response to glucose and IBMX .................................. .. .........

7 . Response to glucose and tGLP- 1 ....................... .. ........... 2 1

C . ln vivo evaluation of encapsulated PTC6-F7 cells ............................... 21 1 . Transplantation studies ........................................................... 21

........................................................... . 1 Glucose tolerance tests 22 3 3 ................................................ ................. . 3 Capsule recovery ., -- 7 7 4 . Immunocytochernis try. ....... .., ................................... --

D . Statistical analysis .............................................................................. 23

IV . Results 24 A . in Vitro Expenmentç ............................................................................

1 . Encapsulated ceIl growth ....................................................... 2 . Insulin secretion fiorn encapsulated cells ............... .... ..... 3 . Response to arginine ................................................................ 4 . Response to sub-physiological levels of glucose ..................... 5 . Response to glucose over 3 1 days ........................................... 6 . Response to ghcose - IBMX ............................... .... ............... 7 . Response to tGLP- I .................................................................

............................................................................. B . in FNo Experiments 1 . Transplantation studies ............................................................ 2 . Glucose tolerance tests ................ ... ........................................ . ...................................................................... 3 Capsulerecovery

V . Discussion 57 A . in Vdro Experiments .............. .. ........................................................ 57

1 . Encapsulated cell growth and insulin secretion ..................... 57 ............................................................. . 2 Response to Arginine 58

3 . Response to sub-physiological levels of glucose ................... 59

4 . Response to glucose over 2 l days ......................................... 59 ................................................. . 5 Response to glucose - IBMX 61

................................................................ . 6 Response to tG LP- I 61

............................................................................. . B ln Vivo Experimenfs 62 ........................................................... . 1 Transplantation snidies 62

.................................................................... . 2 Capsule recovery 63

VI . Conclusion 64

VI I . References 65

List of Abbreviations

E-PTC6-F7:

F-PTC6-F7:

tGLP-I :

HLA:

CAMP:

ER:

G LUT2( f ):

ATP:

ADP:

HIT-Tl 5:

NO:

cGMP:

HbA,C:

UVB:

PL t :

NEDH:

RN:

SV40:

Km:

PTC:

MIN:

STZ:

Encapsulated cells.

Free cells (growing in a monolayer).

GLP- 1 (7-36) amide.

Hurnan Leukocyte Antigen

cyclic- adenosine monophosphate.

endoplasmic reticulum.

Glucose transporter 2 ( I )

adenosine tri-phosphate.

adenosine mono-phosphate.

hamster insuiinorna tumour.

nitrogen oxide.

cycIic-adenosine mono-phosphate.

hemoglobin A, C (glycosylated hemoglobin)

ultraviolet B

po ly-L-lysine

New England Deaconess Hospital

Rat insulinoma

simian virus 40

enzyme equili brium constant.

P-tumour ce11

mouse insulinoma

streptozotocin

Fig. 1:

Fig. la:

Fig. Ib:

Fig. Ic:

Fig. Id:

Fig. 1 e:

Fig. I f :

Fig. 2:

Fig. 3:

Fig. 4:

Fig. 5 :

Fig. 6a:

Fig. 6b:

Fig. 7:

List of Figures

In vitro ceIl growth and insulin secretion by @TC6-F7 cells during the 55 day observation period ..................... .. .. . . . . . . . -. .. . . . . . . .. . - -

Photomicrograph of encapsulated BTC6-F7 immediately after encapsulation ............................... - .......... . . . . . . . . . . . . . . . . . . . . .

Photomicrograph of encapsulated PTC6-F7 cultured in vin-O - 3 days . . . . . . . . -. . . .-. .. . . . . . . . - - . - - . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Page

Photomicrograph of encapsulated PTC6-F7 cultured ii? vitro -7 days.. ..... . . . . . .. . .. ... ... ... . ... .days....................................days.................................... --.. ... .. . .. . .. ... , .. . . .... . ... . .-. . . ... . ... ... . . .- . . . . - - . . . - . . .. . 28

Photomicrograph of encapsulated PTC6-F7 cultured in vitro - 2 1 days . ... .-.. .. ... ... ... ... . .. . .. -.. . .. .... ... ... . . . ... ... . ..... .... ... ...-.........-.. - . . - . . . . . . . . . . . 28

Photomicrograph of encapsulated PTC6-F7 cultured in vitro - 42 days .. . .. . . . . . ... .. . ... .. . .. .... . .. ... ... . ... .. . . . . . . .... ... . ,. ... .... .. . . . . .. . ... .. . . . . . . . . . . . . . . . . . 3 1

Photomicrograph of insulin-imrnunostained of encapsulated ce1 1s cdtured in vitro for 3 1 days ......................................... . . . .. . .. ... .. 33

The effect of arginine ( 1 to 20 mM) on encapsulated and free PTC6- F7 cells ..................... ,... ....... ........ .................................. . . . .. . .. ... -. ..

Response to sub-physiological levels of glucose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... ...... .. ............. ......... . . . . . . .. Cr 37

Glucose responsiveness of encapsulated PTC6-F7 cells over a 2 1- day observation period . . .................................................... . . . .. . . . . . . . 39

Insulin secretion from free and encapsulated cells incubated in O rnM to 16.7 mM glucose in the presence of 1 m M IBMX or IBMX-free media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...--. . . . . . . . . . . . . . . . . . . . . 4 1

Insulin secretion from free and encapsulated PTC6-F7 cells exposed to media containing only glucose (tGLP- 1 control) ..... . . . . . . . . . . . . . . . . . 43

Secretion resulting from the combined effect of glucose and 100 nM tGLP-I on both encapsulated and free PTC6-F7 cells ......... . . .. ... .. . . 43

Summary of encapsulated and free ce11 control incubated in O to 1 6.7mM glucose media. The effect of encapsulated and free cells exposed to 100nM tGLP-I and tGLP-1 free media ....... .... ... ...... ... . ... ... ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . 44

vii

Fig. 8: Blood glucose profiles of STZ diabetic rats following a transplant of 4 .u 10' encapsulated cells .................................... . . . ... . . . . . . . . . - . . . . . . . . .. 47

Fig. 9: PIasma insulin rneasurements of normal (prior to induction of diabetes), diabetic (pre-transplantation) and 4 ': 10' encapsulated cell graft rec ip ients . . . . ....... ... ..... ..... . ... . .. ... ..., .. . . . . . . , . . . . . . . . . . - . . . . . . . . . . . . . .

Fig. 10: Intraperitoneal glucose tolerance tests in 4 n IO' encapsulated ceIl recipients . . . . . . .... ................ ................ ... . . . . . .......... . . . . . . . . . . . .

Fig. 1 1 : Insulin secretion from encapsulated celIs recovered from rats at 30 days post-transplantation ......... ..... ..... .... ... .. ........... ... . . . . . . . . . ..

Fig. 1 ta: Photomicrograph of encapsulated cells recovered from a rat afkr 30 days ... . . ... ... . ... . . ... ... ... . . ..... -.... . . . . . . . . . . . .. . . . .. . .. . . . . . . . . . . . . . . . . . . . .

Fig. 1 I b: Photomicrograph of encapsulated ceils recovered from a rat after 30 day s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,. . . . . . . . . . . . 54

Fig. 1 1 c: Photomicrograph of insulin-irnmunostained of encapsulated cells cultured in vivo for 28 days . .. ........................................ . . . . .. .. . .... 56

viii

I. Introduction 1

1. INTRODUCTION

Diabetes is a condition characterized by metabolic abnomalities leading to

hyperglycemia and long-tenn complications involving the eyes. kidneys. nerves. and

blood vessels '. In insulin dependent diabetes (IDDM), alterations in carbohydrate.

lipid and protein metabolism are caused by the loss of insulin secretion. Insulin and

glucagon are the two main hormones involved in hormonal regulation of blood

glucose levels. Elevated levels of glucose in the post-prandial state tngger the release

of insulin from the pancreatic P-cells. Insulinos main sites of action are hepatic.

muscle and adipose tissue. Insulin. released into portal circulation. acts to stimulate

glycogenesis (in the prandial state) and inhibit hepatic glucose production (in the

fasting state). In muscle and adipose, insulin acts via the insulin receptor that

stimulates glucose transport into peripheral tissues 50. In addition to its primary role

on glucose uptake. various other actions of insulin include: regulation of ion and

amino acid transport. lipid metabolism. gene transcription and mRNA turnover.

protein synthesis and degradation. and DNA synthesis.

A. Diabetes: Etiology

1. Pathogenesis of Tvpe I diabetes

a) Genefic susceptibility: Increasing evidence no w suggests that Type 1

Diabetes (IDDM) is caused by the destruction of the p-cells of the

pancreatic islet by an autoimmune process resulting in deficiency and

finally. a total loss of insulin secretion 3. The first step in the progression

of the disease is susceptibility to IDDM provided by variation in gene(s)

located near or within the HLA (human leukocyte antigens)-encoding

regions of the major histocompatibiiity region. Studies have shown that

association is prirnarily with the alleles of the HLA class 11 (D/DR) loci '. The genetic predisposition, however. is not solely responsible for the P- cel! destruction that ensues.

I. Introduc 1 ion - 7

Environmental trigger event: The second step in the progression of IDDM

remains altogether controversial. A number of factors have been

irnplicated as responsible. Exposure to a virus capable of infecting the P- ce11 has been suggested. Another hypothesis that has generated much

interest is that of bovine albumin (present in cow's milk) resulting in

production of antibodies that also recognize a 69-kDa protein on the

surface of the P-cell. No studies as yet provide any conclusive evidence

Insulitis: The third step in the development of diabetes is the infiltration

of the pancreatic islets by macrophages and activated T lymphocytes.

Activation of autoimmunity: The last stage follows with the conversion of

B-cells fiom self to non-self. This is then followed by the development of

antibodies against a protein present at the P-ce11 membrane surface. The

antibodies may result from the molecular mimicry mechanism descnbed

above. Overt symptoms become present only once more than 90% of the

P-cells have been destroyed.

B. Insulin Secretion and p-cell function:

1. Insulin gene regulation

The insulin gene in al1 species that have been studied is present as a single

copy with the exception of the rat and mouse where there are 2 non-ailelic

insulin genes '". The rat insulin 1 gene differs fiom the rat insulin II gene in a

number of ways. Firstly. the rat insulin II gene. in the absence of enhancer

sequences the promoter is quite strongly active in both insulin-secreting and

non-secreting ce11 lines 5 5 . In addition, the rat insulin I gene has been shonn

to possess a CAMP responsive element which the rat insulin 11 gene lacks j6.

Evidence indicating the presence of negative regulation of the rat insulin II

gene also exists. Mutagenesis (in the rat insulin II gene) of a sequence

corresponding in position to a negative regdatory binding site in the rat

insulin 1 gene results in a two fold increase in the rate of transcription 57.

2. Insulin biosvnthesis

The major control point for acute insulin production is at the level of

translation (glucose stimulates the translation of preproinsulin mRNA within

minutes of exposure). Insulin in its active forrn is a -6 kDa peptide,

consisting of a 2 1 amino acid A-chah and a 30 amino acid B-chain connected

by two interchain disulphide bonds. Briefly. insulin synthesis begins with the

binding of a preproinsulin mRNA to a ribosome present in the cytosol. The

elongation is halted when the signal recognition particle binds to the signal 58.59 recognition sequence (or "pre" chah of the peptide) . This serves to direct

the preprohorrnone/ribosome/mRNA complex toward a signal recognition

receptor (docking protein) on the endoplasrnic reticulum (ER) 60. Upon

binding. the signal sequence releases the signal recognition particle and is

handed over to a second membrane protein. the signal sequence receptor

which facilitates its translocation (through a functional pore) into the ER

where translation into a preproinsulin molecule continues 6 ' . However. during

or shortly after the transfer. the signal sequence is removed by a signal

peptidase. The proinsulin molecules are then passed through the cis. mediai

and tram Golgi cistemae by repeated cycles of budding and fusion. It is in the

tram Golgi network that the secretory vesicles are formed. In the vesicles the

proinsulin is converted to insulin by prohormone convertases (endopetidases).

3. Nutrient-induced insulin secretion

Glucose enten the p-ce11 via the GLUT2 transporter. its subsequent 5 1-52 metabolism results in elevating ATP levels . The rise in ATP:ADP ratios

is thought to inhibit K' channels causing the membrane to depolarize '. This

triggers the opening of ca2' voltage-dependent channels. The influx of ca2-

into the ce11 then ûiggers off the exocytotic mechanisms resulting in the

release of insulin from the B-ce11 setting a cascade of events leading to the

exocytosis of insulin-containing vesicles into the blood.

Studies have s h o w that the initiation of insulin release begins even

before the sudden rise in blood glucose. Oral glucose meals show a

significantly higher insulin response in cornparison to an intravenous glucose

injection. The difference is attributed to the incretin effect. Incretins (such as

glucagon-like peptide- 1. GLP- 1. a 36 arnino acid peptide) released fkom the

intestinal L-ce11 act to potentiate the insulin response in a glucose-dependent

fashion. The other effect of GLP-1 is to inhibit gastric motility thereby

decreasing the rate at which nutrients are absorbed into the blood strearn.

Intracellular CAMP is known to affect glucose -inducecl insulin secretion at 2

levels. A minimal level is required to exert a permissive effect on insulin

release ?'. Cyclic-AMP above this basal threshold can fbrther modulate the

amplitude of the secretory response by changing the sensitivity of the

exocytotic machinery to cytosolic calcium I S .

The metabolism of glucose is clearly the precipitating event by which

glucose-stimulated insulin release rnechanism is enabled (triggered) in the P- cell. The mechanisms involved in amino acid induced insulin secretion.

however. remain to be elucidated. Protein-rich meals and amino acid

infusions are known to elicit an increase in plasma insulin levels 33.3435

Studies comparing the eflect of various arnino acids indicate arginine as

having the strongest effect on insulin secretion 33. Evidence also points to a

synergistic effect between amino acids. This effect rnay also include glucose.

as evidenced by arginine-induced insulin secretion being greater at higher

blood glucose levels in humans 37. Amino acid-stimulated insulin release has

been postulated to act via metabolism of the respective arnino acids.

Cornparison of various studies show that a single pathway by which al1 arnino

acids act remains to be found. Leucine metabolites (a-ketoisocaproate.

isovalerate or acetoacetate) were found to have no effect on insulin secretion

'* . A non-rnetabolizable leucine analogue, BCH (2-amino [2,2,1]-bicylco-

I. Introduction 5

heptane-2-carboxy acid). however. does induce significant increases in insulin

secretion in chlorpropamide-treated dogs 36. Ornithine. an arginine

metabolite, does not stimulate insulin secretion. whereas guanidinoacetic acid

(another arginine metabolite) is a potent stimulator of insulin secretion in the

rat. Oddly this is not true in the dog 39. GPA (guanylpiperadine4-carboxylic

acid). a non-metabolizable arginine analogue does stimulate insulin secretion.

Clearly. leucine and arginine are capable of stimuiating insulin secretion

without being rnetabolized. In the case of arginine-stimulated and GPA-

stimulated insulin secretion. inhibition of glucose utilization (using

mannoheptulose ") results in complete suppression of not only glucose-

stirnulated insulin release but also arginine-stirnulated insulin release ''. This

effect is by no means encompassing of al1 arnino acids. Unexpectedly.

leucine-induced insulin secretion is potentiated by mannoheptulose. Insulin

release induced by arginine appears to require concomitant utilization of

glucose by the P-cells. The glucose dependency of arginine-stimulated insulin

release may in fact act as a safeguard against ingestion of proteins resulting in

fatal hypoglycemia.

Another rnechanism proposes that a signal pathway involving arginine-

denved nitrogen oxide (NO) mediates insulin release fiom isolated rat islets

and HIT-Tl5 cells when stimulated by arginine in the presence of glucose " . The NO is thought to stimulate guanylyl cyclase. leading to an increase in

intracellular cGMP ''4 which has been suggested to be a mediator of insulin

release '". The NO release was reported to be 2-fold greater in the presence of

1 O m M glucose dian without. Inhibition of the enzymatic conversion of L-

arginine to NO resulted in inhibition of insulin release. Maximal activation of

the oxidative pathway in some cells and tissues may be limited by extracellular 46.47 arginine concentrations . Concentration required for maximal uptake in

endothelial cells is within the physiological range of plasma arginine (0.07 to

0.1 1 rnM in humans, 0.1 3 to 0.23 mM in the rat j3). Arginine uptake in islets

is saturated at 15 mM 48. Thus changes in plasma arginine (through diet for

I. Introduction 6

instance) would be expected to regulate the oxidative pathway in B-cells but

not in endothelid cells.

C. Diabetes: Therapy

Blood glucose monitoring followed by exogenous insuiin therapy is the only

route of treatment in IDDM. Conventional therapy usually consists of 2

injections daily where three main types of insulin are used in combination:

regular insulin which acts rapidly with a time to peak of about 2 to 4 hours.

Short-acting (lente or NPH) peak in the range of 6-1 2 hours and the slow-

acting insulin (ultralente) cm 1s t for about 24 hours. Depth and site of

injection can al1 affect the rate of absorption into the blood. hence the time

course of plasma insulin Ievels. Regular insulin is usually given before each

meai (30 min) to provide meal-related insulinemia. The slow-acting insulin is

used to provide basal insulin requirements. The Diabetes Control and

Complications Trial has shown that improved glycemic control by a multiple

daily injection regimen (4 injections per day) can greatly reduce the onset of

secondary symptoms by as much as 70 %. The trade-off to maintaining tight

glycemic control is the 3-fold higher nsk of severe hypoglycemia. ClearIy.

exogenous insulin therapy is not capable of mimicking the closed-loop

feedback between blood glucose and insulin secretion that a fùnctional p-cell

cm accomplish

D. Alternative strategies for insulin replacement in Type I Diabetes

1. M o l e Pancreas transplants

Some studies have show that solid organ pancreas transplants were the on! y

way to achieve a normal glycosylated hemoglobin level (HbAiC is a measure

of long-term glycemic control) and as rnany as 7000 thousand have been done

I. Introduction 7

since 1966 -! A nurnber of problems are associated with pancreas

transplantation. Poor organ availability makes this an impractical option.

Other complications that can arise as a result of whole organ transplants

include: thrombosis. hernorrhage and susceptibility to other illnesses as a

result of intensive immunosuppression required to maintain the foreign

pancreas. Patient morbidity and hospital costs are yet other drawbacks.

2. Isolated islet transplantation

With the advent of islet isolation using collagenase digestion of the exocrine

tissue by Lacy and Kostianovsky the concept of islet transplantation was

introduced ''. The process involves isolating islets from a pancreas. followed

by implantation in a region nch in nutrient and oxygen supply while ensuring

adequate withdrawal of waste metabolites. Transplantation sites that have

been studied include the liver (via intraportai injection 71 -72.73.74.75 ) and lung 'O.

spleen 76. kidney capsule and peritoneal cavity. Additional areas such as the

anterior chamber of the eye. the testis. the brain while purported to be

immunological1y privileged are regions where the immune response simpip

progresses at a slower rate. While syngeneic. intraportal transplants in rats

have been shown to reverse streptozotocin diabetes in rats for the life span of

the animal 79. similar studies in large animal rnodels such as intraperitoneal

transplants in pigs intrasplenic or intraportai studies in primates have met

with limited success. The studies ai1 report a loss of =aft function within a

few days of receiving a transplant. The protection of the islets fiom the

immune systern is thus a key factor for their survival. If the problem of

immunoprotection has been answered. xenotransplantation becomes an

achievable option.

i. Introduction 8

E. Protection from the host's immune systern.

A nurnber of various techniques have been proposed as a means of providing

immunoprotection of islet ph.

1. lmmunomodulation of islet tissue aims at rendering the donor tissue less

immunogenic to the host's immune system. Various strategies for tolerance

induction include culturing islets in 95% oxygen *'. low temperatures 8'.

treatrnent with antibodies to rernove dendritic cells 83 and exposure to W B

irradiation before transplantation. Pre-treatrnent of islets appears to result in

antigen-reactive T-cells becoming indifferent to the transplanted tissue rather

than permanently inactivated 85.

3. Immunosuppression of the host's immune system would expose the recipient

to other dangers resulting fiom a diminished response to foreign invasion.

3 . Irnrnunoisolation allows the islet tissue to be separated from the hast-s

immune system by a semi-permeable membrane which aliows nuniem.

metabolites. O2 and insulin (-6kDa) but impedes passage of antibodies

(-l4OkDa) and imrnunocytes. The matenal (tissue support j and process that

is to be used to irnmobilize the tissue must be biocompatible. The process

must approxirnate physiological conditions as closely as possible to prevent

tissue death or loss of viability. An irnrnunoisolative matenal thus creates a

micro-environment favourable to the donor cells. while not agpvat ing the

recipients' tissues. An effective way of preventing donor tissue rejection

would make islet xenotransplantation a feasible option. hence overcoming the

problem of a readily available source of tissue. The two main types of

irnrnunoisolative techniques that have been utilized are: macroencapsulation

and microencapsulation.

I. Introduction 9

F. Xenotransplantation and Encapsulation.

1. Microenca~sulation: The concept of an encapsulated ce11 that would function

as a replacement for an organ was first proposed by Chang et al 86. The

application of microencapsulation to islet transplantation as a therapy for

diabetes was pioneered by Sun and Lim. who showed for the fint time that

intraperitoneally transplanted microencapsulated islets can reverse diabetes in

animal s 87.90-93.95-98 . This technique has even shown itself to be Feasible in

monkeys 88. The hydrogel nature 89 (85% w/w water) of the alginate-poiy-l-

lysine capsule provides a safe environment for the cells while causing no

imtation to the surrounding tissues. The adoption of an electrostatic

technique to produce the smaller microcapsules greatly improves the capsules'

mass transfer properties 90. The membrane permeability is cntically

dependent on the molecular weight of the poly-L-lysine (PLL) used in fonning

the membrane 94. The larger its molecular weight. the greater the pore size

fomed, hence raising the membrane cutoff barrier. Too low a molecular

weight results in a weak capsule due to less cross-linking between the shorter

PLL chains. The transplantation of microencapsuiated islets requires minimal

surgery. obviating the need for harmful immunosuppressive therapy. The

application of macroencapsulation to islet immunoisolation has s h o w to have

a number of drawbacks. In these devices. islets are contained within a hoilow

fiber membranes made from XM-50 (a p~l~acrylonitrile-polyvinyl chloride

[PAN-PVC] CO-polymer). Upon intraperitoneal implantation these devices

elicited a multi-layer fibrotic pericapsular response ? The devices also result

in the islet tissue clumping together within the wide bore tubular device

resulting in the development of a necrotic core due to decreased nutrient and

0 2 diffusion to the centre. In wide bore tubular devices the access of Oz is

also hampered by the larger difisive distance.

I. Introduction 10

2. Macroencaosulation: Macroencapsulated islets have also been employed as

pefised vascular devices. Artenal blood flows through a semi-penneable

hollow fibre charnber which forms the centre of m u l u s eventuaily emptying

out into the venous system. Arterio-venous (AV) shunts require heavy

anticoagulant therapy to prevent vascular complications. particularly at the

anastomoses. In terms of biocompatibility, di f is ion limitations and the ease

of implantation. therefore. the microcapsule has many advantages over macro-

implants.

G. Cell Therapy

Somatic ce11 gene therapy has been considered as a potential method for

insulin delivery in IDDM 62. Cultured mouse fibroblasts (Ltk3 63. monkey kidney

cells (COS). pituitary AtT20 cells and Chinese hamster ovary cells (CHO) have been

transfected with genes encoding rat and human insulins. The selection and use of

artificially engineered somatic cells as B-ceIl replacements must be viewed with the

following in mind. Initially, the level of gene expression and translation into the

prohormone are key factors in determining the degree to which the insulin production

and storage of the islet p-ce11 will be modeled. The presence of specific

endopeptidases required for processing the proinsulin into insulin limits the choice of

ce11 types. Lastly, regdation of insulin secretion in a manner similar to that of an islet

p-ce11 clearly requires a complex sequence of events based primady on a glucose

handling ability. For instance, while the presence of a specific glucose transponer

may be shown its actual location in a ce11 (cytosolic vs. membrane) may greatly

determine the engineered cells glucose responsiveness.

H. lnsulin secreting &CeIl fines

Insulin-secreting p-cells are endocrine ceils capable of growing in culture.

synthesizing, processing and secreting insulin. Cells of this type are usually derived

I. Introduction 1 1

from induced or spontaneously occurring tumours, transformation of islets using a

viral vector or through transgenic techniques.

1 . lnduced-insulinomas in vivo

X-roy induced Exposure of NEDH rats to x-rays resulted in p-ce11 tumours l 9

(RN-r. -m. -m5f 'O and 1046-38 '' cells. MS-1 30 and -2 cells) that could

be serially propagated in vivo and evennially grown in culture.

BK-virus-induced insulinorna 's: Intracerebral injection of the BK virus into

Syrian hamsters also give nse to pancreatic tumours ". Interestingly.

continuous ce11 lines denved fiom the poorly differentiated Kirkman

hamster insulinoma " did not synthesize insulin in vina but resumed its

synthesis in vivo.

With the exception of the INS cell-line, the ce11 lines thus denved al1 display

functionai irregularities such as a sub-physiological glucose responsiveness '6

20.65 or a complete loss of it which is often combined with a decrease in the

magnitude of the insulin response ? These abnormalities may be correlated

to changes in the cells' glucose metabolisrn at physiological concentrations of

glucose. Other variables include the expression of GLUT-1 transporter in

place of the GLUT-2 (found in normal islets) as their pnmary glucose

transporter. In addition, these cells depict a marked increase in expression of

hexokinase (low Km) rather than glucokinase (high Km) as their primary

glucose phosphorylating enzyme.

2. Transformation of islets in vitro

The HIT-Tl5 ce11 line was produced by an in virro SV40 transformation of

islets isolated fiorn Syrian hamsters. While the transfection of human islets

with a SV40 recombinant vector did result in cells that could be propagated in

virro, it failed to secrete insulin afier 9 passages 66.

I. Introduction 12

3. Insulinomas derived fiom transnenic animals

Equipped with the ability to target expression of oncogenes in particular cells

in transgenic mice. using cell-specific regulatory elements, it is possible to

immortalize rare ce11 types 67. Transgenic mice harboring the insulin-simian

virus 40 tumor antigen hybrid genes (the 5' regulatory region of the rat insulin

II gene is hsed with the early coding regions of SV40 followed by micro-

injection of fertilized mouse embryos) hentably develop p-ce11 tumours that

c m be propagated in culture. The large T-antigen (encoded for by a viral

oncogene in the SV40 genome) binds to the products of two key tumour

suppressor genes of the host cells (Rb and pj3) puaing them out of action.

This releases the cell's safety mechanism that prevents excessive proliferation.

Insulin, therefore. is the ce11 specific marker and T antigen is the hybrid 77 oncogene product. The PTC-1. -2. -3 . -6 '*. -7 '' cell lines and MIN-6. -7 '6

were derived in this fashion. Of these transgenic lines the PTC7-F7 and

MIN-6 lines were found to maintain a correct glucose responsiveness.

Insulinoma ce11 populations consist of a number of ce11 types as shown by

immunohistochemical staining 1 9 . While the presence of al1 3 types of

endocrine cells. with CO-localkation of insulin and glucagon '' have been

shown within the PTC6 ce11 population. insulin. however, is the predorninant

peptide produced. In addition. insulin secretion from the PTC7-F7 clonal line

mimics islet insulin secretion more effectively than the other p-ce11 lines.

Wiîh this in mind, the BTC7-F7 ce11 line thus represents an excellent mode1

for studying the behaviour of a microencapsulated, insulin-secreting cell-line.

both in vitro and in vivo

1. Insulinoma Cell lmmobilization

Recently, the embedded ce11 difision charnber of Miyazaki was used to

immobiIize a mouse insulinoma ceIl line (MlN6) and evaluated for its ability to

I. Introduction

restore normoglycemia in STZ diabetic rats. The diffusion charnben continued to

release insulin for up to 35 days in vitro, and revened hyperglycemia in rats for up to

12 weeks ? Their study also showed the regenerative effect nicotinarnide has on

restoring glucose responsiveness. The nicotinamide is thought to act to restore the

NADH levels in the MIN6 cells which have undergone imrnobilization hence

restoring their ability to cany out oxidative phosphorylation. Nicotinarnide is also

known to have a restorative effect on rats injected with STZ to induce diabetes.

Rat insulinoma cells (RINmSF) cells encapsulated in alginate poly-L-Lysine

capsules when transplanted into diabetic mice were found to reverse hyperglycemia

for up to 7 weeks'" . In addition. encapsulated RMm5F cells were reported to

maintain insulin secretion indefinitely in vitro.

Fragments of a human insulinoma macroencapsulated within tubular Amicon

membranes were reported to have reversed hyperglycemia in streptozotocin rats for

up to 1 year 'O3. The grafts. however. effectively reversed hyperglycemia in 50% of

the graft recipients.

Tumour cells were originally developed as a tool for studying P-ceil function.

Their adaptation as a ce11 therapy obviously requires that following key issues be

addressed for an artificial P-ce11 to fùlfill the requirernents as endogenous insulin-

replacement therapy are descnbed by Efrat in his review 33:

- efficient growth;

- insulin production comparable to norrnal p-cells:

- regulated synthesis & secretion in response to physiological

secretagogues;

- regulatory rnechanism to control cell proliferation when transplanted.

- phenotypically stable.

While meeting al1 these criteria presents no simple task. the question of how

the cells are to be protected fiom the host then arises. Microencapsulation has

demonstrated an ability to protect viable islet cells for as long two years in vivo 88. It

I. Introduction 14

therefore represents a practicaI means of sustaining an artificial P-ce11 to supply

insulin in response to glucose levels. The maintenance of the cells in tems of

protection from the host immune system has been shown. however. it rernains

necessary to study a B-ce11 line behaviour in vitro and in vivo. This is imperative

since the cells cultured in large quantities would be placed in a radically different

environment i.e. from monolayer growth on a flat surface to growth within a

microcapsule. In addition. whether this could have impact on other key criteria such

as ce11 growth rates and insulin synthesis and secretion aiso remains to be secn.

II. Hypo thesis 15

II. Hypothesis

It is proposed that the growth and insulin secretory properties of alginate-

polylysine-alginate (APA) encapsulated PTC-6F7 cells c m be maintained in vifro. It

is also suggested that the APA capsule or encapsulation process has no significantly

deleterious effect on the PTC-6F7 cells' insulin secretory response to glucose. In vivo

the immunoisolative properties of the membrane will mzintain a viable and stable cell

population capable of revening hyperglycemia in swptozotocin diabetic rats while

ensuring protection from the host immune system. The encapsulated cells would.

effectively. provide endogenous insulin replacement therapy.

111 Erperimenral Design & Merhodolo~ 16

III. Experimental Design & Methodology

A. Materials and Methods

1. Ce11 culture

PTC6-F7 cells were grown on 100 mm x 20 mm petri dishes (Falcon) and

passaged approximately once a week. Cells were grown in RPMI 1 640

medium (Tissue Culture and Media Preparation. University of Toronto)

containing 5.5 mM glucose and supplemented with 10% fetal calf serurn

(Gibco). 2 m M L-glutamine (Gibco). 100 U/mL penicillin and sueptornycin

(Gibco). 20 mM HEPES (Sigma) under a 95% air. 5% CO2 atmosphere. The

cells were harvested by incubation with 1.5 mL 0.05% uypsin in 0.53 mM

EDTA (Gibco) for about 2-3 min. to detach them from the petri dish. The

cells were then suspended in 0.9% saline and counted on a hemacytometer

(American Optical).

2. Ce11 encapsulation

The encapsulation technique was a modification of our previously reported

rnethod 9i. The modification involved the use of an electrostatic droplet

eenerator 98 . which produces srnalier. more uniform capsules cornpared to the C

older air-jet technique. The cells were suspended in 1.7% ( d v ) purified

sodium alginate (Kelco Gel LV Kelco. San Diego. CA. USA) at a 6 concentration of approximately 4-8 x 1 O cells per ml. The 5mL cell-alginate

suspension is transferred to a I O mL syringe. Spherical droplets were formed

by the electrostatic field interaction coupled with syinge pump (Razel)

extrusion through a 26G needle and were collecred in a 100 mM calcium

lactate solution. Five to ten minutes later the beads were removed from the

calcium lactate soolution and suspended in 0.05% polylysine (Sigma M. W.

22.000 to 24.000 Da) for six minutes. The droplets were washed with 0.9%

saline and allowed to react with 0.1 j% alginate for 5 minutes. The capsules

were then washed in 0.9% saline before reacting with 55 mM sodium citrate

III. Erperimentd Design & Methodology 17

for 5 minutes. Following a final wash with 0.9% saline and two washes in

culture medium. encapsulated cells were placed in 75 cm2 flasks and cultured

in RPMI 1640 tissue culture medium. On average capsules contained

approximately 60- 100 cells and had an average diarneter of 250-350 Fm.

5. Cell plating

After harvesting and counting the cells (as described above) approxirnately

1x1 o5 cells were added to each well of a 24-weil plate. The cells were then

cultured for 4-6 days under conditions specified above (see ceil cufture

above).

4. Challenge studies

Once the encapsulated cells and fiee cells growing in a monolayer had been

cultured for 4 to 6 days. 200 capsules were hand-picked into each well of a 24

well plate under a dissection microscope using a 3 mL syringe anached to an

18G silicone cannula. The E-PTC6-F7 or F-PTC6-F7 cells were then exposed

to glucose-fiee media for 1 hour to achieve basal insulin secretion. The

glucose-free media was then replaced with media containing the appropriate

secretagogue concentration for a 2 hour period after which a 1 mL sample is

removed and frozen at -20°C for subsequent insulin assay (using a rat insulin

standard. Linco). Media used in challenge studies was serum fiee. The

number of cells per well must then be determined in order to compare the

insulin release fiom cells growing in a monolayer and those immobilized

within the capsule.

5. Ce11 counts

To determine the number of fiee cells per well. 6 wells were trypsinized (see

above) and the cells suspended in 6 mL of 0.9% saline. A sarnple was

withdrawn and a count was performed on a hemacytometer (American

Optical. USA). To determine the nurnber of encapsulated cells per well (by

III fiperimenial Design & Methodology 18

detemining the capsule ce11 density). capsules From 6 wells were collected

and the capsules ruptured to release the celis as foilows; the capsules were

washed twice with 5 mL of 0.9% saline. This was followed by incubation in a

1500 U/mL heparin solution (porcine sodium salt. Sigma) for 10 min at 37°C.

Incubation in a heparin solution results in loss of capsule smoothness and

sphericity. The denatured capsules were washed twice in 5 mL of 0.9% saline.

Two hundred microlitres (200 PL) of 0.05% trypsin in 0.53 mM EDTA

(Gibco) was added to disperse the islet-like ce11 clusters that had formed

within the capsule. The denatured capsules were then ruptured by passing

through 2 1G needle in 1 mL of 50 mM/75 mM sodium citrate/saline. The ce11

suspension was then sampled and only cells that excluded trypan blue were

counted using a hemacytometer (American Optical. USA).

Ilr. Experimental Design & Methodolo~ 19

B. In Vitro hcapsulated pTC6-F7 Cell Response to Secretagogues

In Vitro Ex~erirnent Methodologv

The in vitro insulin response to secretagogues was done to quanti@ the effect

of various known stimulators of insulin secretion from E-PTC6-F7 cells.

Insulin secretion fiom cells growing in a monolayer (fiee cells. F-PTC6-F7)

served as a control for secretion fiom microencapsulated cells (encapsulated

cells, E-PTC6-F7). The fi-ee and encapsulated cells were exposed to basai

media (secretagogue-Free media) for 1 hour to achieve a level of basal

secretion fiom the cells. This was follow-ed by a 2 hour exposure to media

with or without a given concentration of secretagogue(s). At the end of the 2

hour stimulation period, a 1 mL media sample was withdrawn for insulin

assay. Al1 NI vitro experiments were done to measure the insulin response to

each of the following secretagogues: glucose, tGLP-1, IBMX or arginine.

Unless stated othenvise in vitro rxperiments geenerally consisted of four trials

with each trial consisting of three replicate wells.

2. In Vitro Growth and Secretion

The growth of cells within the capsule and insulin secretory response of the

encapsulated cells were assessed over a 55 day penod. At 3-4 day intervals. a

sample of encapsulated cells was withdrawn fiom the culture. Three groups of

a hundred microcapsules were hand-picked under a dissecting microscope and

placed in each well of a multiwell plate (Flow Laboratories, Horsham. PA).

Following a 1 lu pre-incubation in glucose-free medium the cells were

incubated in medium containing 5.5 mM glucose. Two houn later the media

were sampled and fiozen at -209C for subsequent insulin assay. The capsules

were then collected to determine the ce11 density as previously described. In

addition. encapsulated cells at 2 1 days post-encapsulation were fixed in

forrnalin and stained with hematoxylin. To indicate the presence of insulin.

the cells were irnrnunostained with the peroxidase-anti-peroxidase method.

111. Erperimental Design & Methodology 20

3. Response to Arginine

Early BTC6-F7 cells were harvested. one portion of the same batch was

encapsulated while the other plated. The encapsulated and free cells were then

cultured for 1-6 days and pre-incubated as descnbed earlier (see ILA. 1.2.3

and 4 above). Media fiom both the encapsulated and ffee cells were

withdrawn and replaced with glucose-fiee. serum-free media and cultured for

1 hour to bring insulin secretion to basal levels. Afier the 1 hour of glucose-

fiee pre-incubation the F-PTC6-F7 and E-PTC6-F7 cells are then exposed to

media containing the following concentrations of arginine: 0. 1.2.5. 10 or 20

m M arginine. This expenment consisted of a single nial using 12 replicate

wells.

4. Reswnse to sub-~hvsiolog.ical levets of glucose

Encapsulated cells were exposed to sub-physiological glucose concentrations

to determine if the glucose threshold for insulin response had shified to

concentrations less than 4.8 m,M. Cells were culnued. harvested and

encapsulated as described above. Two hundred capsules. each containing

approximately 300 cells were hand-picked into each well of a 24 well plate.

The E-PTC6-F7 cells were then exposed ro glucose-fiee. serum-fiee media for

1 hr. After the one hour pre-incubation. the E-BTC6-F7 were then exposed to

0.0.3. 1. 1.5.2.7 and 16.7 mM glucose media for 2 hours followed bv

sarnpling of the media for subsequent insulin assay.

5. Enca~sulated BTC6-F7 ce11 reswnse to glucose over 2 1 davs

Cells were harvested and encapsulated as described above. The glucose

response of the encapsuiated cells was measured at 7 day intervals over a 2 1

day period. On the day of the challenge. capsules were hand-picked into each

well of a 24-well plate and pre-incubated for 1 h o u (as described in 11.4.4

above). Following the pre-incubation. the media were withdrauii and

111. Erperirnental Design & Methodo& 21

replaced with 0.2.7.5.5 or 16.7 mM glucose media. AAer 2 hour the media

were sarnpled and assayed for insulin. This experiment consisted of a single

trid of 4 replicate wells.

6. Response to glucose and IBMX

After the 1 hour pre-incubation, encapsulated or free cells were exposed to

media containing 1 mM IBMX. in addition to one of the following glucose

concentrations: 0.7.7. 5.5 and 16.7 mM. Control groups were exposed to

glucose only. Media were sampled after 2 hours and assayed for insulin.

Response to glucose - and tGLP- 1

The static challenges were performed as described above (11.A.4. Challenge

studies). These experiments were performed in much the sarne way as the

response to glucose and IBMX (see KB.5 above) with the following

modifications. After the 1 hour pre-incubation the encapsulated or fiee celis

were exposed to media containing 100 nM tGLP- 1. in addition to one of the

following glucose concentrations: 0. 2.7. 5.5 and 16.7 mM. Control groups

were exposed to glucose only.

C. In Vivo Evaluation of Encapsulated pTC6-F7 Cells

In order to study the BTC6-F7 cells function as a P-cell mode1 for insulin-

replacement therapy for diabetes the immunoisolated cells were transplanted into

streptozotocin-induced diabetic rats.

1. Transplantation studies

For the induction of diabetes, Wistar rats (Charles River. St. Constant. PQ)

were administered streptozotocin intravenously at 70 mgkg and after

registenng three consecutive. non-fasting blood glucose measurernent above

111. Eiperimental Design & Merhodology 22

20 m M were considered diabetic and therefore suitable for transplantation.

Twenty-seven anirnals received either an encapsulated or free ce11 graft.

Using light anesthesia 15 anirnals received 4 x 1 o7 cells. 4 received 3 x 107

and 3 received a grafi of 2 x 10' cells. A control group of rats received

corresponding numbers of fiee. non-encapsulated cells. Another control group

received equal numbers of empty capsules. The grafi was administered by

intraperitoneal injection using an 18-gauge cannula. At regular intervals blood

sarnples were taken fiom the tail vein for blood glucose monitoring. To

confirm that the normoglycemic condition of these diabetic anirnals resulted

fiom the graft of the microencapsuIated cells. a pre- and post-transplantation

blood sarnple were taken for insulin determination.

2. Glucose tolerafice tests

Two weeks afier the grafi administration intrapentoneal glucose tolerance

tests (IPGTTs) were administered to transplant recipients in which

normoglycemia had been established as a result of the graft administration.

Diabetic animals were used as controls. In the IPGTT. 1.85 mg glucose/g body

weight in 5 mL of 0.9% saline was injected intraperitoneally into each

experimental rat and blood glucose concentrations were measured at O. 5. 10.

15. 20. 30, 60.90 and 120 minutes later.

3. Ca~sule recoverv

At 30 days post-transplantation. capsules were retrieved by peritoneal lavage

using warm saline. Upon recovery of encapsulated cells From the pentoneum

of the rat at 30 days, 100 capsules were hand-picked into a 24 well plate for

insulin response to 0.7.7.5.5 and 16.7 mM glucose media.

The encapsulated cells recovered fiom the diabetic recipient at 30 days post-

transplantation were fixed in formalin and stained with hematoxylin. In

III. Erperirnenral Design & Methodolo~ 23

addition. to indicate the presence of insulin the recovered cells were

irnmunostained with perioxidase-anti-peroxidase method. The presence of

insulin is revealed by the dark brown colour and intact nuclei are stained by

the blue hematoxylin dye.

D. Statistical analysis

Al1 results are presented as rneans k SEM of the number of values indicated.

Statistical significance was assessed by Student's t test, differences were accepted as

significant at p<O.O5.

/Y Results 24

IV. Results

A. ln Vitro Experiments

1. Encaosulated ce11 growth

The data on in vitro ce11 growth and insulin secretion by PTC6-F7 cells during

the 55 day observation period are sumrnarized in Fig. 1. Within five weeks of

encapsulation. the original density of 30- 1 00 ceIls/capsule increases almost

100-fold. The ce11 density then remains relatively unchanged at around 4000-

5500 cellslcapsuie for the remainder of the 55 day observation period. The

ce11 proliferation within the capsule is shown in Figures la-1 e. At first. the

encapsulated cells proliferated gradually to form islet-like ce1 1 cluster as

shown in Figure Ic. The almost spherioidal cell clusters then continued to

increase in size adopting a less regular shape (Fig. Id). Finally. the ceils

occupy almost al1 of the capsules inner volume as shown in Fig. le. A

photomicrograph of irnmunostaining of encapsulated cells cultured in vitro for

21 days is shown in Fig. 1 f. The dark brown stain for insulin reveals a strong

presence of the peptide CO-locaiized with the light blue stain which reveals the

nuclei of viable cells

2. Insuiin secretion from enca~suiated ceils

Insulin secretion (per 100 capsules) from encapsulated cells ranges From 0.3 to

0.5 ng insulid100 capsules (Fig. 1 ) (2 to 1 1 ng insulin/l o6 cells per hr. graph

not shown) over the first 20 days. During the next 14 days the secretion

increases approximately 10 times to 3.4 ng insulidl 00 capsules after which no

significant change occurs. Taking into account the capsule cell density,

secretion stays at about 5 ng insulinll o6 cells per hr over the same 14 day

penod. Total insulin output expressed per population of cells varies less in

cornparison to expression per population of capsules.

Fig 1 : in Vitro cell growth and insulin secretion by encapsulated pTC6-F7 Cells during the 55 day obsewation period (n=4)

+ Cell density -0- lnsulin secretion

T

O I O 20 30 40 50 60

Time (days)

26 III. Res uhs

Fig. 1 a: Photomicrograph of encapsulated PTC6-F7 irnmediately afier encapsulation. At approx. 100 cells/capsule. Capsule diarneter approx. 3 5 O p .

Fig. 1 b: Photomicropph of encapsulated BTC6-F7 cultured in vitro for 3 days. Small clusters begin to appear. Capsule diameter approx. 350pm-

28 III. Results

Fig. 1 c: Photomicrograph of encapsulated PTC6-F7 cultured in vitro 7 days. Islet-like ce11 clusters are clearly defined. Approx. 500 cells/capsule. Capsule diarneter approx. 3 50pm.

Fig. Id: Photomicrograph of encapsulated BTC6-F7 cultured in vitro 2 1 days. Islet-like ce11 clusters are clearly defined. Approx. 1500 cells/capsule. Capsule diarneter approx. 350pm.

30 III. Results

Fig. le: Photomicrograph of encapsulated PTC6-F7 cultured in vitro 42 days. Islet-Iike ce11 clusten are clearly defined. Approx. 1500 ceIls/capsule. Capsule diameter approx. 3 5 0 p

32 ZII. Results

Fig. 1 f: Photomicrograph of insulin-immunostained of encapsulated cells cultured Nt vitro for 2 1 days. The dark brown stain for insulin reveals a strong presence of the insulin CO-localized with the light blue stain which reveals the nuclei of viable cells.

3. Response to Arginine

The insulin responses of encapsulated and free cells to O. 1.2. 5. 10 and 20

mM concentrations of arginine are shown in Fig. 2. Overall. the secretion

pattern from encapsulated cells is similar to the secretion from cells growing

in a monolayer. Insulin secretion from BTC-6F7 cells does not change

significantly over the O mM to 5 mM arginine concentrations but rarher varies

between 8- 15 ng insulin/l o6 cells per hr. At 10 mM arginine, fiee cells release

almost 6-fold more insulin as compared to the O to 5 m M arginine range (54.4

I 7.9 ng insulidl o6 cells per hr. p<0.05). The encapsulated cells at 1 O mM

arginine, however. release only about half as rnuch insulin as do the fiee ceils

at 10 rnM arginine (p<0.05). Exposure of fkee cells to 20 mM arginine results

in a secretion of approxirnately 86.6 t 13.8 ng insulin/l o6 cells per hr which is

comparable to 8 1.2 f 13.2 ng insulinIl o6 cells per hr released fiom the

encapsuiated cells.

Fig. 2: The effect of arginine on encapsulated and free pTC6-F7 cells This experiment consists of a single trial of 12 replicate wells.

1 I I Free cells 1 / Encapsulated Cells /

Arginine (mM)

4. Response to sub-ghysio1oe;ical levels of glucose

The results of the PTC6-F7 response to low glucose levels are s h o w in Fig. 3.

A sub-physiological response pattern entails an increase in secretion over the

low concentrations of glucose (€4-5 rnM) followed by the curve reaching a

plateau over the higher (>5 mM) glucose concentrations beyond which no

M e r changes in secretion are observed. In effect the threshold for glucose-

stimulated insulin secretion is reduced. The insulin response From

encapsulated cells to sub-physiological levels of glucose is somewhat blunted

as shown in Fig. 3. In general. secretion ranges from 12 to 17 ng insulin/106

cells per hr. Almost no change in secretion is observed between O and 1 mM

glucose. Insulin secretion is significantly higher at 1 SmM glucose (1 7.1 I 1.4

ng insulidl06 cells per hr . p<0.10) as compared to exposure of the

encapsulated cells to glucose-fiee medium (12.0 + 1.2 ng insulin/1O6 cells per

hr). However as the glucose concentration is increased to 16.7 m M there is no

change in the insulin release. The secretion fiom the PTC6-F7cells therefore

portrays a lack of glucose responsiveness.

Fig. 3: Response to sub-physiological levels of glucose (n=4).

0.0 0.3 1 .O 1.5 2.7 16.7

Glucose (mM)

IV. Results 38

5 . Res~onse to glucose over 21 davs

The results of the encapsulated cells' glucose response over 2 1 days is shown

in Fig. 4. The response pattern to glucose over the 2 1 day observation period

remains relatively unchanged. in that maximal secretion tends to occur at 16.7

mM glucose. On the day of encapsulation, the secretion over the O to 5.5 m M

glucose range varies between 3-7 ng insulin/106 cells per hr while high

glucose ( 16.7mM) elicits a secretion almost 2.5-fold that at the O to 5mM

range. The magnitude of stimulation (the "fold" increase in insulin secretion

induced by 16.7mM glucose does not change much over the 2 1 day study

period. The arnount of insulin release per ce11 does undergo notable change at

14 days post-encapsulation. Secretion at 14 days in the O to 5mM media

glucose range is over 3-fold greater as compared to secretion at the respective

glucose concentration range at O and 7 days post-encapsulation. At 2 1 days

the secretion at al1 glucose media values are slightly decreased in comparison

to secretion at 14 days however remain 3-fold as compared to that

immediately post-encapsulation.

Fig. 4: Glucose responsiveness of encapsulated PTC6-F7 cells over a 21day observation period. This experiment consists of a single trial of 4 replicate wells.

Glucose (mM)

= O I I 2.7

5.5 16.7

O 7 14 21

Time (days)

6. Response to glucose + IBMX

The presence of 1 rnM IBMX in the medium results in an increased insulin

response to glucose as shown in Fig. 5. Insulin secretion fiorn encapsulated

and fiee cells were sirnilar when exposed to the sarne media. Exposure to

glucose-fiee. IBMX-fiee media results in insulin secretion of about 8-1 2 ng

insulin/l06 cells per hr. Exposure to 2.7 mM glucose media results in a 3.5-

fold increase in insulin secretion as compared to exposure to glucose-fiee.

IBMX- free media (p<0.05). Insulin secretion remains essentiall y unchanged.

ranging from 33 to 43 ng insulinA06 cells per hr. over the 2.7-1 6.7 m M

glucose range without the presence of 1 mM IBMX. In the presence of

glucose-free media however. 1 mM IBMX induces an almost 3.5-fold release

of insulin over IBMX-fiee media. While ImM IBMX significantly potentiates

insulin secretion it does not greatly alter the pattern of release. A maximal

secretion of 162- 182 ng insulidl06 cells per hr at 5.5 mM glucose remains

unchanged in spite of increasing the glucose concentration to 16.7 mM. The

effect of glucose alone on the cells results in a maximal secretion at 1.7 mM

glucose. whereas in the presence of I m M IBMX maximal secretion is

realized at 5.5 mM glucose.

Fig. 5: Insulin secretion from free and encapsulated cells exposed to O - 20 mM glucose with or without the presence of 1 mM IBMX.

Free Encap. 0 Free + 1 mM IBMX

Encap. + ImM IBMX

O 2 5 20

Glucose (mM)

7. Response to tGLP-1

Insulin secretion fiom free and encapsulated cells exposed to glucose-only

media is shown in Fig. 6 a while secretion resulting fiom the combined effecr

of glucose and 100 nM tGLP-1 is s h o w in Fig. 6b. Secretion from both Free

and encapsulated cells remains steady at about 10 to 15 ng insuiid1 o6 cells

per hr over the O to 16.7 mM glucose media range. Secretion fiorn

encapsulated cells in the presence of 5.5 mM glucose was almost 2-fold as

cornpared to that in glucose-fiee media. This response. however. was not

rnaintained at higher levels of glucose. In the presence of 100 nM tGLP- 1

both free and encapsulated cells maintain a sirnilar. stepwise increase in

insulin secretion. Increasing the media glucose concentration fiom O rnM to

5.5 mM. in the presence of tGLP-1. results in raising secretion from

encapsulated cells and fiee cells about 1.5-fold and 3-fold respectively.

Further increasing the media glucose to 16.7 m l (while mainraining 1 O O a !

tGLP- 1 ) only succeeds in restoring insulin output to levels obsened from

exposure to media containing 100nM tGLP- 1 and no glucose. A surnrnary of

the data described above are showri in Fig. 7. Both fiee and encapsulated cells

when exposed to glucose-fiee media with 1 OQn!! rGLP-1 result in an almost

2-fold increase in insulin secretion over glucose-Free controls. While both

encapsulated and fiee cells dcpict a suonger secretion of insulin in the

presence of tGLP-1. the Free cells secrere about 15% more insulin at 2.7 and

5.5 rnM glucose. The insulin secretion fiom both the experimentai (tGLP-1

stimulated) and control fiee cells showed a decrease at hi& glucose ( 16.7

mM) when compared to secretion at 5.5 mM glucose.

Fig. 6a: Insulin secretion from free and encapsulated BTCG-Fi cells exposed to media containing only glucose.

35

I I Free cells - 30 - Encapsulated cellç

Glucose (mM)

Fig. 6b: lnsulin secretion from free and enmpsulated cells exposed to glucose in the presence of 100 nM tGLP-1.

Glucose (mM)

Fig. 7: Summary of Fig. 6a and 6b. Data for al14 expenmental groups are shown. Encapsulated cells or free cells are exposed to glucose (O to 16.7 mM) in the presence of 100 nM tGLP-1. Control groups receive no tGLP-1.

i Free i

60 ; = Encapsulated ! il Free + 100 nM tGLP-1 1 i Encapsulated + 100 nM tGLP-1 1

0.0 2.7 5.5 16.7

Glucose (mM)

B. ln Vivo Experiments

1. Trans~lantation studies

The table below sumrnarîzes the results of the ce11 transplantation studies in

Wistar rats. The average blood glucose profile of recipients of 40 million

encapsulated cells are shown in Fig. 8 (arrows indicate the number of animals

whose blood sugars have no! reverted to hyperglycemia).

Type of # of # of # of rats that Duration of PTC6-F7 cells txp. graft recipients underwent reversal of ce11 grafi reversal of hyperglycemia

hyperglycemia (davs) Encapsulated 40 million 15 12 22 + 5 cells: 30 million 4 3 16 I 3

20 million 3 1 5

Free cells: 40 million 2 O - 30 million - 7 O - 20 million 1 O -

Total # of graft recipients: 27

The effect of the ce11 transplant is detected at grafts greater than 30 million

cells. The average duration of the reversai of diabetes extends at least an

additional 6 days on average when 40 million encapsulated cells are

transplanted. Random blood glucose measurements Vary between 2 and 6 mM

over the duration of the graft. Of the 15 animals receiving 40 million cells 2

died of severe hypoglycemia while a third showed no amelioration in

hyperglycemia. An intraperitoneal transplant of fiee cells results in a partial

lowering of blood glucose levels which in general remain far above normal.

h i m a l s receiving an equivalent volume of empty capsules show no change in

blood glucose concentrations. The plasma insulin (see Fig. 9.) of grafi

recipients (2.5 5 0.3 ng insulinlml) showed a 4-fold increase over the nomial

rats (0.6 f 0.1 ng insuiidml prior to induction of diabetes) and 40 times that

of the diabetic rats (0.06 + 0.02 nglinsulin).

Fig. 8: Blood Glucose Profiles of STZ diabetic rats following a 7

transplant of 4 x 10 Encapsulated pTC6-F7 cells.

Txp 4x1 ~~enca~sulated cells

O 10 20 30 40 50 60 70

Time (days)

Fig. 9: Plasma insulin measurernents of normal (prior to induction of diabetes), diabetic (pre-transplantation) and 4 x 1 o7 encapsulated cell graft recipients (n=4).

Normal Dia betic P re-txp.

Dia betic Post-txp.

IV. Results 49

2. Glucose tolerance tests

Results of the intraperitoneal glucose tolerance tests are s h o w in Figure. 10.

The graft recipients had random blood glucose levels in the range o f 2-4 mM.

Intraperïtoneal injection of glucose resulted in rapid rise in blood glucose over

the tirst 20 min to just over 5 mM in grafi recipients as compared to a peak of

1 OmM observed in normal rats. This was followed by a decrease to 5 rnM at

60 min post-injection and to a final value of 3.5 mM at 2 hours.

Fig. 10: lntraperitoneal glucose tolerance test

in 4x1 0' encapsulated cell recipients (n=4).

\ Diabetic (Pre-txp.)

'--3

Diabetic (Post-txp.) - *--- -A O 20 40 60 80 100 120 140

Time (min)

IV. Results 5 1

3. Capsule recovew

lnsulin secretion From capsules recovered by an intraperitoneal lavage with

saline and subjected to a glucose challenge are shown in Fig. 1 1 . Secretion

ranges from 1 to 2 ng insulin/lo6 cells per hr. Photomicrographs of recovered

encapsulated cells are shown in Fig. 1 1 a and 1 1 b. The capsules recovered

from the diabetic graft recipients tended to contain an amorphous material

surrounding the ce11 clusters (Fig. 1 la). In addition insulin immunostaining

revealed the presence of insulin CO-localized with viable cells. however there

is also a fair amount of insulin in the region surrounding the ce11 cluster (see

Fig. 1 lc).

Fig . 1 1 : lnsulin secretion frorn encapsulated BTC6-F7 cells recovered at 30 days post-transplantation (n=l )

2.7 5.5

Glucose (mM)

53 111. Results

Fig. 1 la: Photomicropph of encapsulated cells recovered from a rat afier 30 days.

Fig. 1 l b: Photornicrograph of encapsulated cells recovered fiom a rat d e r 30 days. Note the amorphous material surrounding the ceIl cluster at the centre.

Fig. 1 1 c: Photomicropph of insulin-irnmunostained of encapsulated cells culniled in vivo for 3 0 dais. The dark brown stain reveals the presence o f insulin. Viable cells are revealed by the blus hematoxylin stain.

K Discussion 57

V. Discussion

In this study. the in vitro growth of encapsulated BTC6-F7 cells and insulin

secretion by the encapsulated cells is evaluated over a 55 day period. In addition. the

Nt vitro insulin response of the encapsulated cells to glucose. tGLP- 1. IBMX and

arginine is also investigated. The objective to try and use these cells to reverse

hyperglycemia in streptozotocin-induced diabetic rats also represents an important

milestone in showing that a replicating p-ce11 does in fact perform well in providing

endogenous insulin replacement therapy.

A. In Vitro Experiments

1. Encapsulated ce11 growth and insulin secretion.

In the growth and secretion experiments, insulin secretion from encapsulated

cells shows little change over the first 20 days of culture while the ce11 density

increases 35-fold (see Fig. I ) . The microcapsule serves as an effective barrier

to limit ce11 growth to a mass of cells that fil1 the inner capacity of the

microcapsule. Capsule rupture due to the "pressure'? of cells growing within is

not observed even when capsules are completely full (see Fig. le).

Microencapsuiation of the cells results in a radical change in their

environment; from monolayer growth on a flat-surfaced petn dish to radial.

three dimensional growth within a hydrogel inside a capsule. Hydrophiliciw

of the immunoisolative matenal has been shown to affect ce11 growth within

synthetic polymen. The hydrogel nature of the alginate capsule clearly

provides an environment suitable for cell proliferation even for an attachent-

dependent cell. Hence. the lag in insulin secretion may be attributed to the

cells' adaptation to growth in their new surroundings. This observation is in

keeping with the data previously published by Miyazaki et al ". In their

expenments mouse insulinoma cells (MIN6) were immobilized in an agarose-

PVMA (polyvinylmethacry1ate)-collagen matrix and cultured for up to 35

days. While little or no change in insulin accumulation into the medium was

K Discussion 58

observed over the first 21 days an approximately 5-fold increase occurred over

the next 14 days ". In the data shown (Fig. 1 ), at approximately 55 days when

the ceil numbers appeared to have stabilized, the insulin secretion is found to

be about 10-fold greater compared to the secretion at 19 days. The growth

curve and insulin secret ion pattern demonstrate that cells immunoisoiated

within an APA capsule are capable of maintaining their physiological

cornpetence over the entire observation penod. In theory, the development of

a necrotic core is of concem. particularly when the growth of cells in

aggregates may present a difisive banier to the entry of nutrients and

withdrawai of waste products 'O1 . The histologicai sections of the encapsulated

cells fixed at 21 days (Fig. 1 f) indicate a large fraction of viable cells (as

determined by the incorporation of the blue hematoxylin stain which highlights

the presence of intact nuclei) at the ce11 aggregate core. Core necrosis is thus

not a significant problem. In addition. the necrotic cores described by

Sutherland result in aggregates of up 5 x 1 o4 cells which f o m 600pm

spheroids. The microencapsulated cells fonn aggregates of up to 5000 cells

with a diameter less than 300pm.

2. Response to Arginine

In the data s h o w (see Fig. 2), the BTC6-F7 ce11 insulin response does

not become apparent until a threshold of 10 m M arginine is reached. The

threshold rnay thus lie between 5 and 10 mM arginine. The discrepancy

between the fiee and encapsulated ce11 insulin secretion at 10 rnM arginine

remains anomalous because there is no significant difference in secretion

between encapsulated and fiee cells in the presence of 20 mM arginine.

Physiological plasma levels of arginine range between 0.07-0.1 1 m M in

humans and 0.1 to 0.2 mM in rats j3. Uptake of arginine by normal. islet P- cells is saturated at 15mM arginine 48. Exposure to a 1 OO-fold greater

concentration of arginine may in fact be resulting in ce11 death and non-specific

release of insulin stored within the cells. Previous studies have shown that the

l? Discussion 59

arginine-induced insulin release by normal P-cells is CO-dependent on glucose

utilization 34. 35.37 . If the glucose handling mechanism in the B turnour ce11 is

disrupted, as will be discussed later, then it may also result in an abnormal

arginine response. A lower threshold for glucose may be linked to a decreased

threshold for arginine-induced insulin secretion as well.

3. Response to sub-phvsioloe;ical levels of glucose

A lefi-shifi in the glucose-responsiveness of the PTC-6F7 cells impiies that

concentrations of glucose below that of physiological levels (<1 mM) induce

increases in insulin release fi-om the cells. The 40% increase in the secretion

at 1.5 mM glucose (Fig. 3) as compared to O mM glucose, while significant

(~'0.05). is not enough evidence to suppon the notion that the cells have

assmed a '-sub-physiological response". This is primarily because the

secretion at glucose concentrations higher than 1.5 rnM (2.7 and 16.7 mM)

rather than remaining at the secretion induced at 1.5 mM decreuses to the

levels induced by glucose-fiee media. The insulin response of the

encapsulated cells remains insensitive to glucose-only media as shown in Fig.

3. Rather, the data shows that the PTC6-F7 ce11 line exhibits a constitutive

release of insulin regardless of the glucose concentration.

4. Response to glucose over 2 1 davs

Investigation of the glucose responsiveness of encapsulated and cultured over a

21 day period indicates that the responsiveness is rnaintained to a minimal

degree of consistency (see Fig. 4). The low insulin output from the cells when

exposed to high glucose medium on Day O may be due to some cells

recovering from the encapsulation procedure. On Day 7 the secretion is only

slightly lower and one rnay speculate that the encapsulated cells may be

undergoing the process of adapting to their new environment and undergoing

growth. This is not unlike the observations made in the growth and secretion

experiments discussed below. By Days 14 and 21 the encapsulated cells have

?! Discussion 60

stabilized at the 10-25 ng insulin /106 cells per hour secretion rate normally

exhibited in vitro.

Glucose-responsiveness represents a key discnminating factor between nimour

P-cells and normal p-cells. In these expenments. (with the exception of the glucose

response observed over a 2 1 day period shown in Fig. 4). exposure to high glucose

( 16.7 mM) resulted in virtually no change in insulin secretion over glucose-fiee

medium (Fig. 3, Fig. 5 and Fig. 7). In this respect. the PTC-6F7 cells exhibit a

response that is very unlike that of isolated pancreatic islets. In his review. Malaisse

showed a s ipoidal insulin response curve using isolated rat islets exposed to various

glucose concentrations. Extrapolation of the response data taking into account the

basal (0-2.5 mM) glucose media release of 17 uU insulidisiet per 90 min penod

(about 80- 100 ng insulid 1 o6 cells per hour assuming about 3 500-5000 P-cells/islet)

and a maximal release of about 320 uU insulin/islet per 90 min (1 500-2500 ng

insulin/l o6 cells per hour) at 20 mM glucose. shows that the threshoid level for

glucose-stimulated insulin secretion is 4.8 mM glucose. Thus the data show that the

BTC-6F7 cells differ fiom isolated islets in a number of ways: i) Basal secretion and

the magnitude change in response to glucose stimulation is low; ii) The cells have a

lower threshold for glucose-induced insulin secretion. Glucose phosphorylation. by

high-Km glucokinase. in islets is now accepted as the glucose "sensor" in the p-cell.

Lack of glucose responsiveness has been attributed to changes in the

glucokinase/hexokinase ratios ".

The PTC-6F7 cells were initially cultured in glucose concentrations of 25 mM.

This level of glucose was deemed necessary to maintain their responsiveness 49.

Exposure to physiologically relevant levels of glucose may have resulted in changes in

the glycolytic enzyme ratios present in the cells. The continuous exposure of the cells

to a constant 5.5mM glucose level may result in the glucose transport mechanism to

operate optimally at this concentration and hence result in a desensitization to glucose

concentrations higher than 5mM. This may explain why certain passages of cells

exhibit a maximal insulin output at 5.5mM glucose (see Fig. 6a. and 6b.). This is not

V. Discussion 61

always the case though as seen in Fig. 4. Other changes which are certainly harder to

attribute ba i s to are the consequences of placing the cellos insulin promoter under

control of a viral expression vector. One of these factors may include the --pre-

occupation" of the cells to undergo division rather than synthesize insulin.

Raising levels of cAMP using IBMX or tGLP-1 appears to have improved the

magnitude of the insulin response. As will be discussed below, the "sensitizing"

ability of CAMP in improving insulin secretion is very apparent.

5. Response to glucose + IBMX

Isobutylmethylxanthine (IBMX) indirectly raises intracellular cAMP levels by

phosphodiesterase inhibition hence preventing its breakdown. The elevated

levels of CAMP act to increase glucose uptake by the ce11 which is the primary

step leading to insulin release (see 1.8.3). It is interesting to note that IBMX

potentiates insulin release fiom BTC-6F7 cells even when no gZucose is

present (see Fig. 5). This aspect of CAMP'S effect is further discussed below.

The overall effect of IBMX is thus to simply potentiate the release of insulin

From the cells as shown in Fig. 5.

6. Response to tGLP-I

Glucagon-like peptide (7-36 amide. tGLP-1) is a potent hormonal mediator of

the entero-insular axis involved in the regulation of glucose homeostasis 1 8 .

This incretin peptide also represents a more physiological secretagogue when

compared to IBMX especially in light of its role as an insulinotropic agent.

Studies in PTC-I cells have shown tGLP-1 to be able to stimulate insulin

biosynthesis 1 8 . Glucagon-1 ike peptide- 1 binding to a G-protein coupled

receptor results in adenylate cyclase activation, hence leading to an elevation 1+ 18 of intracellular cAMP leveis and Ca- . The increase in CAMP levels occurs

without the presence of glucose 19. The elevation in cAMP is required to

mediate the gluco-incretin effect of tGLP-1 i.e. the potentiation of insulin

secretion in the presence of stimulatory concentrations of glucose ". The

K Discussion 62

presence of tGLP- 1 clearly restores a certain degree of glucose dependent

insulin secretion (see Fig. 6b and Fig. 7). This improvement in the glucose-

responsiveness in P-cells with elevated cAMP levels correlates well with the

study done by Wang et al who showed restoration of glucose responsiveness in

j3-cells purified by fluorescence activated ce11 sorting 68. In the experiments

descri bed above. the PTC6-F7 cells maintain insulin secretion even in the

absence of any secretagogues including glucose. Glucagon-like peptide4

appears to sensitize the insulin release mechanism. Thorens et al have shown

that elevation of cAMP leading to activation of protein kinase A sensitizes the

GLUTZ transporter 69. The 2-fold increase in insulin secretion in giucose-free

medium supplemented wiih tGLP-1 (see Fig. 7) is not surprising if the cells'

sub-physiological glucose response is considered. The direct effect of cAMP

on the insulin secretory machinery of the BTC6-F7 cell may be more

pronounced than in normal cells. Comparison of the cells' response to glucose

over the 2 1 day study (Fig. 4) and the tGLP- 1 study (Fig. 7) indicate a certain

degree of inconsistency. Insulin secretion at high glucose appears to be

inhibited by 16.7mM glucose and peaks at 55mM glucose in the tGLP-1

experirnents. In the 2 1 -day response experiment. however. secretion is

maximum at 16.7mM glucose. This variation can only be attnbuted to the

difference in cell passage.

B. In Vivo Experiments

1. Transplantation studies

The intraperitoneal implantation of a suficient number of encapsulated PTC6-

F7 cells (>30 million) into diabetic animals results in reversal of diabetes

within 24 hours. Continuous insulin secretion with liale glucose

responsiveness results in hypoglycemia as was the case with the 2 ,4 x 10' ce11

graft recipients which succumbed to dangerously low blood glucose. While

there is no data on whether the rats were hyperphagic. withdrawal of food

resulted in transplant recipients often becorning hypoglycemic. The markedly

Bat glucose clearance curve (see Fig. 10) clearly reflects the steady supply of

insulin with linle regard for blood glucose concentrations. Graft recipients had

high levels of plasma insulin (see Fig. 9) hence the initial peak in blood

glucose barely rises above normal. While the growth of cells within the

capsules represents an important factor in the reversai of hyperglycemia in rats.

on the other hand, continuous proliferation within the capsule may result in

dangerously low blood glucose levels. Streptozotocin-induced diabetic rats

require high doses of insulin (toronto or lente and ultralente) (3-4U/day for a

300-350 g rat) and yet glycemic control remains poor. In addition. variation in

severity of diabetes for instance can result in the same dose of cells reversing

blood glucose in one animal while having littie effect in another. The fact that

hyperglycemic rats are reversed by the grafis appears paradoxical in that the

grafis produce much less insulin than the arnount required by the rats if

provided by injection. It may be worth considering the way in which the

insulin was delivered (i.e. continuous release h m the graft into portal

circulation vs. a bolus dose given subcutaneously)

2. Capsule recoverv

The insulin response to glucose. while significantly lower in cornparison to in

vitro data, in combination with positive immunostaining for insulin in

encapsulated cells recovered fkom the diabetic. grafi recipient Further confirms

their physiological functionaiity during the entire observation period (see Fig.

1 I ). The strong staining for insulin surrounding the ce11 cluster within the

capsule (Fig. I Lc). in addition to the arnorphous material surrounding a similar

cluster in Fig. 1 1 a, suggests the presence of insulin-containing cellular debris.

VI.

VI. Conclus ion 64

Conclusion In vifro. microencapsulated cells continue to proliferate and secrete insulin

h m within the microcapsule. This clearly demonstrates the lack of any deleterious

effects that the APA microcapsule environment or the microencapsulation process

may have on BTC6-F7 ce11 growth and insulin. Insulin secretion from

microencapsulated BTC6-F7 cells in response to glucose is potentiated by tGLP-1 and

IBMX. agents which both act to elevate intracellular CAMP leveis. In addition. once

the capsule is full. the membrane wall serves as an effective barrier to lirnit ce11

growth.

While the growth of microencapsulated PTC6-F7 cells in vivo appears to be

weaker in cornparison to growth in vitro. the reversai of diabetic hyperglycemia in 14

out of 15 streptozotocin-induced diabetic rats represents an important milestone in

demonstrating the efficacy of microencapsulated ce11 therapy for insulin dependent

diabetes.

There remain however a number of issues with regards to the criteria

previously listed (see introduction. p. 14) that require M e r investigation. A high

level of insulin production is important in that fewer cells (and hence fewer capsules)

would be required to reverse hyperglycemia. Achieving this would involve

transfection with the insulin gene. followed by over-expression. ideally resulting in

increased insulin biosynthesis. Lower graft volume would ensure better long temi

function in vivo. in addition. the regulation of insulin secretion is fundamental to

avoid the dangers of hypoglycemia. It rnay be possible to manipulate the expression

of the lower Km hexokinase to which glucose non-responsiveness is atuibuted.

Genetically engineered and transgenically derived cells may be a feasible

alternative to isolated pancreatic islets as a source of insulin-secreting tissue-

However. their ability to maintain these properties fiom within a permselective

membrane is a crucial step towards their applicability in ce11 therapy. The growth and

insulin secretory properties of microencapsulated PTC6-F7 cells provide an excellent

mode1 of insulin-secreting cells as a means of providing endogenous insulin

replacement therapy.

VIL Rejerences 65

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