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t1 OCR I nde x As A Ra pid Po te nc y Me tric o f I so la te d I sle ts o f L a ng e rha ns By Chris F ra ke r Unive rsity o f Mia mi Sc ho o l o f Me dic ine OBS: How it works OBS: How it works An oxyg e n se nsitive Ruthe nium - -

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SLIDE 1

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OCR I nde x As A Ra pid Po te nc y Me tric o f I so la te d I sle ts o f L a ng e rha ns By Chris F ra ke r Unive rsity o f Mia mi Sc ho o l o f Me dic ine

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SLIDE 2

OBS: How it works OBS: How it works

2

  • Consumption by c e lls
  • Diffusion a c ross ma trix
  • O 2 - que nc he d Ru(DPP)3Cl2
  • Diffusion from a ir into we ll a nd me dia

An oxyg e n se nsitive Ruthe nium An oxyg e n se nsitive Ruthe nium -

  • ba se d

ba se d fluor

  • phor

e fluor

  • phor

e is adsor be d to silic a pa rtic le s is adsor be d to silic a pa rtic le s a nd e mbe dde d in a silic one rubbe r ma trix a nd e mbe dde d in a silic one rubbe r ma trix

2 2

Cells Cells

t2

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SLIDE 3

ME T HODS:

Oxyg e n c o nc e ntra tio n is c a lc ula te d using mo difie d e q ua tio nso f Ste rn-Vo lme r T he o ry

Model Ster

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 0.00 0.05 Normalized Relative Fluorescence or Io / I

n-Volmer Behavior

0.10 0.15 0.20 [O2] (atm) Io / I Fluorescence normalized to that at 0.21 atm

I 0 / I A = 1 + KSV [ O2] A

t3 t6

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SLIDE 4

ME T HODS:

Oxyg e n c o nc e ntra tio n is c a lc ula te d using mo difie d e q ua tio ns o f Ste rn-Vo lme r T he o ry

  • I

0 / I A is the dyna mic ra ng e of the pla te fluore sc e nc e sig na l, DR

  • K SV is the Ste rn Volme r c onsta nt de fine d a s (DR- 1)/ [O 2 ] A
  • F

luore sc e nc e va lue s for e a c h we ll we re norma lize d (by division) to the ir initia l va lue a nd the n to the a ve ra g e va lue of no- c e ll c ontrols a t e a c h time point (NRF )

  • Ra tio of DR/ NRF

= I 0 / I

NRF

  • DR/ NRF

= 1 + K SV * [O 2 ]NRF

  • So, NRF

= DR/ (1 + K SV * [O 2 ]NRF )

  • F

luore sc e nc e va lue s so- norma lize d we re use d to c ompute oxyg e n c onc e ntra tions pe r E qua tion [1]. DR 1 [O2]NRF

= — — —

x — — — [1] NRF

  • 1

K

sv

t3 t6

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SLIDE 5

T ra nsie nt Sta te dO 2 Slope a nd E quilibrium OCR Ca lc ula tions

Shor t Shor t-

  • te rm (< ~1 hr)

te rm (< ~1 hr)

  • Qua ntita te b a se d o n ra te o f c ha ng e

Qua ntita te b a se d o n ra te o f c ha ng e

  • f sig na l
  • f sig na l
  • T

ra nsie nt dO T ra nsie nt dO 2

2 Slo pe ~ Slo pe o f 1

Slo pe ~ Slo pe o f 1-

  • 1/ NRF

1/ NRF

  • vs. time
  • vs. time
  • I

de a l fo r I sle ts I de a l fo r I sle ts

Ste a dy Sta te Ste a dy Sta te

  • Qua ntita te d

Qua ntita te d b a se d o n e q uilib rium OBS b a se d o n e q uilib rium OBS sig na l sig na l

  • OCR = D S L

∆p / h

  • OCR ~ Ac tua l 1

OCR ~ Ac tua l 1-

  • 1/ NRF

~ DdO 1/ NRF ~ DdO 2

2

  • Additio na l

Additio na l c o mpa ritive c o mpa ritive me tric o f isle t me tric o f isle t po te nc y. po te nc y. “ “ T rue OCR T rue OCR” ”

  • T

ime to e q uilib rium de pe nds o n T ime to e q uilib rium de pe nds o n vo lume o f me dium use d. vo lume o f me dium use d. T = h T = h2

2/ D

/ D

D = 3.3 x 10- 5 c m 2/ s S = 0.31 c m2 for a 96- we ll pla te t4 h = 0.65 c m for 200 µL L= 6.0 x 107 L / c m3 * fmol/ µmol * s/ min

∆p= in µM.

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SLIDE 6

ME T HODS: T riplic a te we lls o f I E Qs in L

  • w (2.2mM) a nd

Hig h (22mM )Gluc o se Supple me nte d K re b s Buffe r

Islets Low glucose Islets High glucose Media CNT Sulfite CNT

  • Me a sure fluore sc e nc e c ha ng e for 200

c yc le s 5 minute inte rva ls

  • Ca lc ula te tra nsie nt sta te dO2 a nd e quilibrium OCR

a nd norma lize for DNA c onte nt.

  • Inde x = Me a n Hig h g luc ose va lue s/ Me a n L
  • w g luc ose va lue s

t3 t6

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SLIDE 7

E xa mple Asse ssme nt: Isle t Shipme nt F rom UPS/ Se a ttle 6- 29- 05

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Se a tlle Ship m e nt # 3 6 - 2 9 - 0 5 5 0 1 0 0 1 5 0 2 0 0 2 5 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 Tim e (m inute s) O 2 (uM ) 5 0 0 Lo w 1 5 0 0 Lo w 2 5 0 0 Lo w 3 5 0 0 Hig h 1 5 0 0 Hig h 2 5 0 0 Hig h 3 Ra p id d O 2 Eq uilib rium

T ra nsie nt Pe rio d I nde x: 1.51 E q uilib rium I nde x: 1.56

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SLIDE 8

T r ansie nt pe r iod dO2 slope and E quilibr ium OCR sc ale kine tic ally with c e ll numbe r

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Cell T itr a tion Hum a n I E Qs 50 100 150 200 250 200 400 600 800 1000 T im e (m inutes) dO2 (uM) 500 250 125

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SLIDE 9

E quilibr ium OCR

  • vs. I

E Q #

y = 310.59x - 23856 R

2 = 0.99

y = 404.97x - 18497 R

2 = 0.9949

40000 80000 120000 160000 200000 200 400 600 I E Q # E quilibrium OCR (fmol/ min) L

  • w

Hig h

dO 2 a nd E quilibrium OCR Sc a le L ine a rly with IE Q #

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SLIDE 10

Both me tric s of OCR sc a le with IE Q a nd e a c h othe r

Rapid dO2/ DNA vs. Equlibrium OCR/ DNA y = 124928x + 1059.9 R2 = 0.8509 0.0E+00 1.0E+04 2.0E+04 3.0E+04 4.0E+04 5.0E+04 0.0E+00 5.0E- 02 1.0E- 01 1.5E- 01 2.0E- 01 2.5E- 01 3.0E- 01 Rapid dO2/ DNA (uM/ min/ ug DNA) Eqm OCR/ DNA (fmol/ min/ ugDNA)

Ra pid dO2 ra w vs. Equilibrium OCR ra w

y = 108337x + 18084 R2 = 0.811 0.0E+00 5.0E+04 1.0E+05 1.5E+05 2.0E+05 2.5E+05 0.0E+00 5.0E- 01 1.0E+00 1.5E+00 2.0E+00 Rapid dO2 (uM/ min) Eqm OCR (fmol/ min)

t9

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SLIDE 11

Dia be tic Mouse Mode l for Pote nc y

  • Che mic a lly Induc e d Dia be tic Mic e
  • Isle ts T

ra nspla nte d into Kidne y Ca psule

  • Re c ipie nts c hose n ba se d on body

we ig ht a nd blood g luc ose profile similar itie s.

  • 2000 IE

Q pe r mouse

  • Monitor

blood gluc ose daily

  • Cure is de fine d a s two c onse c utive da ys

< 200 mg / dl fa sting blood g luc ose

  • Da ys to c ure use d a s c ompa ra tive me a sure
  • f pote nc y.

t11 t12

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SLIDE 12

OCR/ DNA shows some c or r e lation to r e ve r sal…

T ra nsient sta te dO2 slope vs. Reversa l Ra te 0.00 0.20 0.40 0.60 0.80 1.00 1.20 0.000 0.200 0.400 0.600 0.800 1.000 T ra nsient sta te dO2 slope KRB Hig h (uM/ min/ ug DNA) T ra nspla nt Suc c e ss Ra te

t13

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SLIDE 13

...but OCR Inde x is muc h more pre dic tive

solid red = pelleted --> all failed

  • pen red = not pelleted, but failed at least some replicates

yellow = 100% reversal, but not all by day 3 green = 100% reversal by day 3

  • pen green: n=1

* asterisk = PFC

t13

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SLIDE 14

Ka pla n- Me ie r type surviva l a na lysis.

  • P- va lue s c a lc ula te d using a log - ra nk te st. P < .00001
  • T
  • p c urve “c ure ” c urve for mic e with ra tios > 1.27
  • Bottom c urve , mic e with ra tios < 1.27.
  • Da she d line s 95% c onfide nc e inte rva ls for the fra c tion c ure d.

t15

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SLIDE 15

In 31 huma n isle t pre pa ra tions a na lyze d, sta tistic a l a na lysis be a rs out tha t inde x is by fa r the be st pre dic tor

t14

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SLIDE 16

Ac knowle dg e me nts:

BD

  • Ma rk T

immins, PhD.

  • Ric ha rd D. Gua rino , MS.
  • Sha ro n Pre sne ll, PhD.
  • Pe rry D Ha a la nd, PhD.

Unive rsity of Minne sota Dia be te s Institute for Immunolog y a nd T ra nspla nt

  • K

le a rc ho s Pa pa s, PhD.

Unive rsity of Mia mi Dia be te s Re se a rc h Institute

  • Da ma ris Mo la no , DVM
  • Anto ne llo Pile g g i, MD
  • Hiro hito I

c hii, MD, PhD.

  • L

uc a I nve ra rdi, MD

  • Ra fa e lla Po g g io li, MD
  • E

lsie Za hr

  • Jill Co llins
  • Ca millo Ric o rdi, MD