OCR I nde x As A Ra pid Po te nc y Me tric o f I so la te d I - PowerPoint PPT Presentation

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 - - ba se d ba se d An oxyg e n se nsitive Ruthe nium fluor ophor e is adsor is adsor be d to silic a pa rtic le s fluor ophor e 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 t2 • Diffusion from a ir into we ll a nd me dia 2 2 • Consumption by c e lls Cells Cells • Diffusion a c ross ma trix 2 • O 2 - que nc he d Ru(DPP) 3 Cl 2

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 n-Volmer Behavior 6.0 5.5 Fluorescence or Io / I 5.0 Normalized Relative 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.00 0.05 0.10 0.15 0.20 t3 [O2] (atm) t6 Io / I Fluorescence normalized to that at 0.21 atm I 0 / I A = 1 + K SV [ O 2 ] A

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]. t3 t6 DR 1 [O2] NRF = — — — x — — — [1] NRF - 1 K sv

T ra nsie nt Sta te dO 2 Slope a nd E quilibrium OCR Ca lc ula tions 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 Shor Shor t- t - te rm (< ~1 hr) te rm (< ~1 hr) � ∆ p / h OCR = D S L � 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 t4 o f sig na l o f sig na l � OCR ~ Ac tua l 1 � OCR ~ Ac tua l 1- -1/ NRF 1/ NRF ~ DdO 2 ~ DdO 2 � T � T ra nsie nt dO 2 Slo pe ~ Slo pe o f 1- -1/ NRF 1/ NRF ra nsie nt dO 2 Slo pe ~ Slo pe o f 1 � 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 vs. time vs. time po te nc y. “ po te nc y. “ T T rue OCR” rue OCR ” � T � T ime to e q uilib rium de pe nds o n � I ime to e q uilib rium de pe nds o n � I de a l fo r I sle ts de a l fo r I sle ts vo lume o f me dium use d. vo lume o f me dium use d. 2 / D = h 2 T T = h / D D = 3.3 x 10- 5 c m 2 / s S = 0.31 c m2 for a 96- we ll pla te h = 0.65 c m for 200 µL L= 6.0 x 107 L / c m3 * fmol/ µmol * s/ min ∆ p= in µM.

ME T HODS: T riplic a te we lls o f I E Qs in L o 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 t3 t6 • 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 ow g luc ose va lue s

E xa mple Asse ssme nt: Isle t Shipme nt F rom UPS/ Se a ttle 6- 29- 05 t10 Se a tlle Ship m e nt # 3 6 - 2 9 - 0 5 2 5 0 Ra p id d O 2 Eq uilib rium 2 0 0 1 5 0 O 2 (uM ) 5 0 0 Lo w 1 1 0 0 5 0 0 Lo w 2 5 0 0 Lo w 3 5 0 0 Hig h 1 5 0 5 0 0 Hig h 2 5 0 0 Hig h 3 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 Tim e (m inute s) T ra nsie nt Pe rio d I nde x: 1.51 E q uilib rium I nde x: 1.56

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 t7 Cell T itr a tion Hum a n I E Qs 250 200 150 dO2 (uM) 500 250 125 100 50 0 0 200 400 600 800 1000 T im e (m inutes)

dO 2 a nd E quilibrium OCR Sc a le L ine a rly with IE Q # E quilibr ium OCR vs. I E Q # 200000 quilibrium OCR (fmol/ min) y = 404.97x - 18497 160000 t8 2 = 0.9949 R 120000 L ow Hig h 80000 y = 310.59x - 23856 2 = 0.99 40000 R E 0 0 200 400 600 I E Q #

Both me tric s of OCR sc a le with IE Q a nd e a c h othe r t9 Ra pid dO2 ra w vs. Equilibrium OCR ra w Rapid dO2/ DNA vs. Equlibrium OCR/ DNA 2.5E+05 5.0E+04 Eqm OCR (fmol/ min) (fmol/ min/ ugDNA) y = 124928x + 1059.9 2.0E+05 Eqm OCR/ DNA 4.0E+04 R 2 = 0.8509 3.0E+04 1.5E+05 1.0E+05 2.0E+04 1.0E+04 5.0E+04 y = 108337x + 18084 R 2 = 0.811 0.0E+00 0.0E+00 0.0E+00 5.0E- 01 1.0E+00 1.5E+00 2.0E+00 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) Rapid dO2 (uM/ min)

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 t11 • 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 t12 of pote nc y.

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 1.20 1.00 t13 ra nspla nt Suc c e ss Ra te 0.80 0.60 0.40 T 0.20 0.00 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)

...but OCR Inde x is muc h more pre dic tive t13 solid red = pelleted --> all failed open red = not pelleted, but failed at least some replicates yellow = 100% reversal, but not all by day 3 green = 100% reversal by day 3 open green: n=1 * asterisk = PFC

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 t15 • T op 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.

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

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