EX-VIVO SLAUGHTERHOUSE

PORCINE CRYSTALLOID-PERFUSED

BEATING HEART VIA LANGENDORFF

METHOD

By: Rahiemin Talukder Advisor: Dr. Ali N. Azadani

UC Denver Biology BS University of Denver ENBI Bioengineering MS

MME Department

1

PRESENTATION STRUCTURE Introduction and

Background

Heart Transplantation

Anatomy

Physiology

Langendorff system

Preservation

Hypothermia

Reperfusion

Previous Studies

Materials and Methods

Harvesting

Preservation

Resuscitation

Results

Discussion

Additives, Limitations

Future Research

Developments

Conclusion

2

ORGAN TRANSPLANTATION

Figure 1 – OrganDonor.gov Organ donors and

recipients and in-waiting in the US[2]

3

Roughly 29,000

transplantations

were performed in

2014.

3,965 are

awaiting

heart transplants.

In 2014,

2174 heart

transplants

were performed

in the US.

BACKGROUND

4

HEART ANATOMY

5

Figure 2 - Standard anatomy of the heart with

deoxygenated blood from body circulation towards the

lungs on the right side of heart and oxygenated blood

from lungs pumped towards systemic circulation[15]

CORONARY CIRCULATION OF THE HEART

Figure 3- anatomy of coronary circulation of

arterial (left) and venous (right) vessels [11]

As the heart works in vivo, coronary flow occurs during ventricular

diastole periods 6

CARDIAC CELL MEMBRANE PHYSIOLOGY

Table 2 – average

extracellular vs intracellular

composition[13]

Ion Equilibrium

Potential

Potassium -90 mV

Sodium +67 mV

Calcium +123 mV

Chloride -86 mV

Table 3 – Equilibrium potential of core

ions in cell [13]

Extracellular

concentration

(mM)

Intracellular

concentration (mM)

Na+

K+

Ca2+

Cl-

Mg2+

ATP

glucose

145

4

1

110

1.5

0

5.6

15

150

4

10

17

4

1

7

ACTION POTENTIAL

Fast response

Photo Credit: http://www.pathophys.org/physiology-of-cardiac-

conduction-and-contractility

Figure 5 – (left) Action potential in myocytes. The fast response organizes near

simultaneous contractions between muscle cells

(right) slow response is carried out by pacemaker cells 8

Figure 4 - Resting membrane

potential Photo credit:

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/ExcitableCells.htmL

Resting state of cell

FAST RESPONSE CARDIAC ACTION POTENTIAL

9

Outside cell

Inside cell

Na+ channel Ca2+ channel K+ channel

ECG BASED ON GLOBAL ACTION POTENTIAL

Figure 6 Depolarization propagated waves of

both atrial and ventricular function

corresponding into ECG readings using 3

leads on a body. ECG on isolated heart will

differ Photo credit: (http://www.clicktocurecancer.info/vascular-resistance/cellular-

cardiac-electrophysiology.htmL)

10

Figure 7 - ECG

video with cardiac

electrical

conduction

correspondence

ISOLATED HEART PERFUSION

Langendorff technique is used to resuscitate isolated hearts

Neuronal and hormonal regulation eliminated

To have the heart resuscitated, it must be preserved and then reperfused

This technique is standard for heart transplantations Figure 8 – Oscar Langendorff 11

Photo credit: http://iphyspc12.med.uni-rostock.de/hist/langendorff.htm

LANGENDORFF TECHNIQUE

Figure 9 - Aortic valve function

and location in aortic root

12

Oxygenated retrograde flow

administered towards the aorta,

causing the aortic valve to shut.

Flow is then redirected towards

the coronary ostia and into the

coronary vasculature, with

unoxygenated venous return

exits out of the coronary sinus

into the right atrium

Photo credit: www.encyclopedia.lubopitko-bg.com

LANGENDORFF REPERFUSION MODES

Brought by pressured

columns and

compliance chamber

Driven by gravity

Autoregulation with

oxygen uptake

Too high pressure can

let on towards edema

perfusion via roller

pump

Constant Pressure Constant Flow

13

PIG AND HUMAN SIMILARITIES Adult

Human

Adult

Pig

Average body

weight (kg)

62-71 86

Average heart

weight (g)

250-350 358

Resting heart

rate (bpm)

60-100 100 -

150

Average

temperature (C⁰)

37 39

Similar anatomy of four

chambers, weight, and

orientation

Xenotransplantation

performed in past but led

to fatal results due to

immunologic responses to

the host body

Table 4 – Human (left) and Pig (right) physiological comparison

14

Photo credit: http://funmozar.com/real-human-heart/

PRESERVATION: CARDIOPLEGIA (HEART-PARALYSIS)

15

• Solution that enables electromechanical cardiac

arrest

• Extracellular solutions mimic blood (Na+)

• Intracellular mimics inside cell composition (K+)

• Plegisol is extracellular

• Cost-effective

• UW solution is intracellular and has additives

that increases preservation effectiveness

osmotic support

adenosine to increase ATP storage

antioxidants, etc

HYPOTHERMIA/METABOLISM

Von’t Hoff principle: Oxygen uptake consumption reduces 50%

for every 10C decrease in physiological temperature

Metabolic rate is decreased, mechanical arrest has occurred and

thus ATP production is ceased and stored

Figure 9 – Logarithmic values displayed for metabolism of endothermic

species with respect to temperature (1000/K) values [55].

16

-10 C 60C 20C

REPERFUSION

Must be at 37C and pH of 7.4 and oxygenated at 9-11 psi

Blood Red blood cells as oxygen carrier

must always contain an anti-coagulant.

Flow rate at 3-4 mL/min/g

Krebs Henseleit Buffer (KHB) Transparent for visual purposes

Similar to the ionic concentration of blood composed of salts and glucose

Direct oxygenation into solution

Flow rate at 8-12 mL/min/g

Calcium must be 1-2.5 mM. Too much will cause for less compliancy due to edema while too less won’t enable contractions

Diluted blood with KHB Many studies take advantage of hemoglobins in red blood cells and

proteins while using KHB at 1:4 blood-KHB 17

PREVIOUS STUDIES

18

These studies show that similar cases are working for 4 hours with oxygenated reperfusion at 37C

Discover missing points from patents of previous studies enable us to discover how to develop a working Langendorff system at DU

EINDHOVEN UNIVERSITY OF TECHNOLOGY LIFETEC

BIOHEART

Slaughterhouse pigs are used

Reperfusion: Mixed KHB and

filtered heparinized blood

collected from another pig.

Hematocrit at 25%

3 L circulating perfusate

Hearts resuscitated for 4

hours with stabilized sinus

rhythm

19

PRELIMINARY STUDIES – VISIBLE HEART LAB [33]

Langendorff schematic of the Visible Heart Lab pertaining human and pig hearts [27]

Heart is excised in lab; transport & set-up time 5 minutes

Rewarming stage is 30 minutes

Crystalloid perfusion only

Video and camera quality image due to transparent solution

Work was patented and not all details provided providing us to discover and fill in the gaps

20

MATERIAL AND METHODS

21

• Harvesting

• Preservation

• Reperfusion

HARVESTING

120-kg swine of Yorkshire,

Berkshire, and Hampshire breed

are brain-dead and

exsanguination occurs, starting

warm ischemic time (WIT).

WIT is kept 1.5 minutes – 7

minutes. WIT is detrimental to

heart survival. Other

slaughterhouses had longer WIT

Excised hearts must be beating

so that cardioplegia is the factor

that arrests and not cell death

WIT ends when cold ischemia is

initiated by hypothermic topical

saline with ice slurry to wash all

blood

22

Figure 10 – Trimming tissue

from excised heart

PRESERVATION Cannula is inserted, tied,

and clamped into aorta

1-L of 4C cardioplegia

coronary flush enables aortic

valve shut for solution to

redirect towards coronary

vasculature

Aortic valve must be checked

to be shut

Heart is placed in sealed

transport bag (1-L 4C UW or

Plegisol) submerged in ice

1-hour Transport time

Cold ischemia lasts 1.5-2

hours

23

Figure 11 – Cannulated heart with aortic valve shut under 72 mmHg

CORONARY CATHETERIZATION

Figure 14 – DLP®/Gundry ® Retrograde Coronary Sinus

Perfusion cannula with manual-Inflate Cuff

Figure 13 – Medtronic DLP ®

Multiple Perfusion Set

24

Figure 12 – Cannula insert

APPARATUS

25

Millipure water for buffers and cleaning

7 L modified Krebs Henseleit Buffer

2.1 g/L Sodium bicarbonate

Calcium chloride dihydrate (1.5-2.5

mMol/L)

Mannitol (2.92 g/L )

Heparin (5K-15K/L )

Insulin (10 U/L)

20KU Streptokinase/heart

Epinephrine (0.25 mL/L)

Carbogen tank for oxygenation and

carbon dioxide to stabilize pH to 7.4 at

9-11 psi

Water heater

Perfusion chamber

Pressure transducer

Air bubble trap

Thermometer gun

ZOLL ® R-series defibrillator and ECG

Single-Chamber Medtronic pacemaker

Figure 15 – DU Langendorff

schematic

RESUSCITATION Slow rewarming of heart

before cannulated into Langendorff system. Most recently-excised heart is the first subjected tested to keep its cold ischemic time as low as possible

Defibrillator applied when arrhythmia occurs at 15-30 Joules, at least 1 minute intervals

Pacing at rate 100 ppm, output at 15 mA, and sensitivity at 2.5 mV

26 Figure 16 – Single Chamber Medtronic

Pacemaker

Photo credit: http://www.m-e-t.co.za/shop/cardiac-rhythm/medtronic-5348-single-chamber-temporary-pacemaker-3/

RESULTS

27

SETTINGS AND MODIFICATIONS FOR LANGENDORFF-PERFUSED ISOLATED HEARTS Test Experimen

t Date

Experime

nt heart

test

(n = )

Constant

Flow (CF)

or Constant

Pressure

(CP)

Cardioplegi

a additives

(Heparin

(H) or

Streptokin

ase (SK)

t = 0 hour

(peak avg)

(bpm)

T = 1/2 hour (peak

avg) (bpm)

1 Aug 20 1 CP H 52

(appendage

only)

0

2 CP H 78(append

age only)

0

2 Sept 24 3 CP H 54

(appendage

only)

30

4 CP H 0

3 Oct 8 5 CF H AFib 0

6 CF H N/A 0

4 Feb 16 7 CF H 0

8 CF H 0

5 May 6 9 CF H 86 VF

10 CF H AFib 0

6 July 8 11 CF H & SK N/A 0

12 CF SK N/A 0

28

Test Experiment

Date

Experime

nt heart

test

(n = )

Constant

Flow (CF) or

Constant

Pressure

(CP)

Cardioplegi

a additives

(Heparin

(H) or

Streptokina

se (SK)

t = 0 hour

(peak avg)

(bpm)

T = 1/2 hour

(peak avg)

(bpm)

7 Nov 4 13 CF SK 120 0

14 CF SK 30 160

8 Jan 27 15 CF SK 145 79

16 CF SK 0

9 Feb 3 17 CF SK 100 0

18 CF SK 60 0

10 Feb 17 19 CF SK 100-180 0

20 CF SK 150-180 0

11 Mar 24 21 CF SK 176 24

22 CF SK 0 0

12 Mar 31 23 CF SK 143 126

24 CF SK 185 58

13 April 7 25 CF SK 172 110

26 CF SK 147 92

29

SETTINGS AND MODIFICATIONS FOR LANGENDORFF-PERFUSED ISOLATED HEARTS

RESULTS

Results are determined by the following:

Heart rate mimicking human standards of 60-120 bpm

ECG recordings for stabilized QRS complex

contractile function recovery

Aortic pressure of 80-120 mmHg

30

TEST 1, N = 1

Heart applied via constant pressure

Langendorff perfusion with cannula

attached towards aorta for n = 1

Saline was 0-1C

Plegisol is pH of 3-4

Appendage beating for only 15

minutes

31

Figure 17

Figure 18

TEST 5, N = 9

Plegisol is modified with

sodium bicarbonate,

stabilizing pH to 7.4 and

activating ingredients

Atrial contractions occur

before fibrillating.

Epinephrine is

administered

Ventricular contraction

not seen due to coronary

blockage

32

Figure 19

TEST 5, N = 9

33

Figure 20 - Artery shows KHB

perfusion but other minor

vessels and veins show blood

unable to pass through

HEPARIN VS STREPTOKINASE

Heparin is an

anticoagulant used for

blood and serves for

preventative action

Streptokinase is a

thrombolytic agent for

thrombi established in

vessels that prevent

coronary flow specifically

in the usage of the heart Figure 21 (Cossum) Coagulation

pathways of plasma factors

34

ENZYMATIC KINETIC ACTIVITY – PH AND TEMPERATURE

Figure 22 a & b –

enzymatic activity effected

by pH and temperature

35

TEST 7, N = 13

SK/HEP-INDUCED CARDIOPLEGIA PRESERVATION

0.176 g CaCl2

WIT is 6 min

500 mL of 20KU SK and 15KU heparin –infused tepid saline flushed prior to cold cardioplegia

Constant flow reperfusion mode

Pacemaker sensitivity set to ASYNC to initiate pulse instead of augmenting SA-driven pulse

Atrial conduction for 30 min

Torsade de Pointes: R on T is visible

Ventricular/apical contraction still not occuring n = 13 initiated global atrial

contraction with HR 30. Atrial

tachycardia approaches before

fibrillation occurs

36

Figure 23

TEST 8, N = 15

Figure 24 a & b – ECG readings at key observance of definite

fibrillation with 240 HR, proceeding fibrillation with HR 90, and

observance of polymorphic ventricular tachycardia, Torsade de

Pointes

37

*ECG will not

Follow a regular

3-lead ECG taken

From a human

Body as the heart

Is isolated

TEST 8, N = 16

Figure 25 – Fibrillation can

be determined by the second

inclined wave in the QRS

portfolio ( n = 16) and HR 260

Figure 26 dictates the

‘normal’ heart rate due for n =

16

38

TEST 11 – PRESSURE TRANSDUCER UTILIZED

Figure 27 – Pressure transducer usage in schematic

39

Figure 28 – Pressure

Transducer and air

trap

*Oscillations due to roller pump.

Mean aortic pressure resided in

80-120 mmHg, following physiological

range

MODIFICATIONS FOR TEST 13 PROCEDURE

Excised beating hearts with lung block intact were immediately submerged in cold saline.

WIT 3-4 minutes

Ostia catheterization of 100 mL 20KU SK-infused room temperature Plegisol applied at a time to ensure both arteries had flow

Waste saline is changed by cold fresh saline

While tepid cardioplegia in cold saline submergence, hearts were still beating

Lab: Reperfusate (KHB) has 2.0 mM CaCl2/L

*Hearts had highest rate of compliancy than all previous tests

Hearts defibrillated at 20 J til stabilized. 0.25 mL/L Epinephrine is administrated prior to defibrillation

Needle prick would let air emboli escape from coronary arteries

40

TEST 13, N= 25

Figure 29 – Anterior Langendorff apparatus and set-up for n = 25

41

TEST 13

42

43

TEST 13, N = 25 ECG

Figure 31 – Vtach at HR 110 (top) and stabilized ECG reading for n =

25 at HR 65 with aortic pressure at 74.7 mmHg

44

DISCUSSION

45

• Studies have been done to resuscitate the Langendorff work but

details aren’t always provided. Our lab ran 26 experiments learning

of details not provided

• Modified procedures to increase global contractile function

• HIGHLIGHTS:

• Excised heart is beating

• WIT

• Cooling techniques and changing used saline

• Streptokinase-infused room temperature cardioplegia

• catheterization

• Temperature and rewarming

• Defibrillation until stabilization

• no blood clots

• No air emboli

LIMITATIONS

WIT Animal Lab and seeking out

dog hearts at CSU

Equipment

Data acquisition Pressure-volume

Left ventricular end-diastole pressure

Residing Defibrillator/ECG

Sterilization (autoclave)

Apparatus for only 1 heart at a time (increases CIT for 2nd heart)

Foam production

Edema Less compliancy

Leads to organ dysfunction

Equipment and measurement of edema to moderate its significance

46

Figure 32 – Foam accumulation

occurred for all tests

ADDITIVES TO LANGENDORFF

Perfluorocarbons

Enables improved oxygen uptake in crystalloid solutions mimicking that of hemoglobins on RBC

Blood

To increase oxidation and protein utilization aiding cardiac performance

Heparin for animal lab

47

FUTURE WORKS AS A PLATFORM

Comparing donor human hearts unable to be

used for transplant to pig hearts

Collaborating with local hospitals to test original

devices and aid in their studies

National Jewish Center is interested in testing dog

hearts with MRI. We can use a mobile Langendorff

system

Anschutz Medical Campus is interested in Ablation

therapy

Experimentally validate TAV flow and

hemodynamic parameters, and ventricular assist

device testing

48

WORKING-HEART MODEL

Chamber Pressure (mmHg)

Preload RA 2-10 mmHg

LA 5-15 mmHg

Afterload RV 10-20

LV 60-80

Table – Loading pressure constants

for cardiac chambers mimicking

physiological values

Figure 33 – Proposed

schematic for

Langendorff/4-chamber

convertible apparatus

49

MOBILE LANGENDORFF

Faster accessibility and usage by eliminating

transport and thus cold ischemic time

Considerations:

Oxygen tank

Electrical outlets

Sterilized environment and access to all equipment,

water heater

50

TRANSMEDIC ORGAN CARE SYSTEM: HEART ™

Improved patient outcomes;

Increased utilization of available organs;

Expanded supply of organs; and

Reduced total cost of care.

Key functions:

• Physiological Monitoring

• Blood Oxygenation

• Warming

• Pulsatile Flow

51 Figure 34

Credit: https://www.youtube.com/watch?v=MZxRTYs-dyk

CONCLUSION

Our work’s primarily focus is to utilized the

beating heart platform for vast research in

biomedical device applications and heart

transplantation methods.

This work is created in hope that it educates a

more-detailed procedure for those seeking to

initiate Langendorff-perfused isolated hearts for

research applications at DU

52

RESEARCH ADVENTURES

53

THANK YOU!!

Thank you to my committee:

Dr. James Fogleman, Dr. Matthew Gordon, Dr. Breigh Roszelle, Dr. Ali

Azadani

Dr. Corinne Lengsfeld, Dr. Mohammad Matin

Innovative Foods LLC

Emanual, Pepe, Patti, Diane, and Dave for being flexible, meeting our

research needs and keeping WIT 3-5 min.

Outreach for aid

Dr. Dr. Chris Orton at Colorado State University

Dr. Ashok Babu at Anschutz Medical Center

Mathieu Poirier and Cory Wagg at University of Alberta

Ron Richards of President of Rocky Mountain Perfusionists Inc,

Dr. Balsingham Murugaverl of DU’s chemistry department

DU’s Professional Research Opportunities for Faculty fund at the University of

Denver.

Lab Partners: Alex Clinkenbeard & Benjamin Stewart

And always, thank you to my parents and brother Ryan for their sacrifices they’ve

made along with me, keeping me going when I wanted to give up, driving me to work

hard and try first, and do good second.

54

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57

Appendix

58

ST. THOMAS HOSPITAL #2 (PLEGISOL)

Plegisol is an efficient cardioplegia with hyperkalemic properties

that mechanically arrests the heart.

More efficient at higher temperatures compared to other

cardioplegia such as UW solution

This solution is cost-effective

Table – Composition of Plegisol Solution

59

UNIVERSITY OF WISCONSIN (VIASPAN)

UW solution is optimal for long-term

preservation cold storage past 6 hours,

evidently leading to better graft

outcomes

Potassium lactobionate: Osmotic

support, decrease cell swelling

KH2PO4: Potassium source

MgSO4: Desiccant (drying agent on

how water interacts)

Raffinose: Hypertonicity for cell

desiccation before cooling

Adenosine: Reduces ATP catabolism

rate, inhibits platelet aggregation &

inflammatory cells, decreases

superoxides, increases contracture

Glutathione: Reducing agent, oxidizes

free radicals

Allopurinol: Lowers uric acid in blood

plasma

Hydroxyethyl starch: Prevents shock

based on blood loss

Table -

60

IONIC CONCENTRATIONS OF ALL SOLUTIONS In Vitro

(mmol)

Plegisol

(extracellula

r)

(mmol)

UW

(Intracellular)

(mmol)

KHB

(extracellular)

(mmol)

In

cell

Out of

cell

Na+ 5-15 150 120 30 142.5

K+ 150 5 16 120 4.4

Cl- 160.4 - 126.7

Ca2+ 10-4 2.5 1.2 - 1.76

Mg2+ 16 5 1.2

H2PO4 - 25 1.2

SO4 - 5 1.2

HCO3 10 - 25

Osmolality 300 315 290

pH 7.8 7.4 7.4

61

CALCIUM AND CALCIUM OVERLOAD

Physiologically, calcium is at 1.4-2.5 mMol/L. However, half

of that is ionized while the other half is bound to protein

which is unavailable in the crystalloid solution

Calcium overload can lead to noncompliant rigorness due

to:

Edema – Water is drawn into the mitochondria,

hampering ATP productivity

High Na+ gradient – as Na+ diffuses abnormally higher

intracellularly, Na+ is pumped back out, and more Ca2+

pumped back in

Lactate formation

Reperfusion-induced myocardial injury via oxidative

stress 62

STREPTOKINASE DOSAGE IN PATIENTS

Rout

e

Dosage/Duration

I.V.

infusio

n

1,500,000 IU/60

min

I.C.

infusio

n

20,000 IU (bolus)

2,000-4,000 IU/min for

30-90 min (60 min

average)

Table Streptokinase dosage

for IC and IV in clinical

settings

Author Organ Dosage Effect

Szyrach,

2011

Porcine

kidneys

12,500 U/L

vs. 50,000

U/L

50KU led to

toxic effects

Stark, 1988 heart 25000

U/100 mL

Faster

sinus node

recovery

time

Mickelson,

1988

Rabbit

heart

150 U/mL LV function

recovered

and kept

LVEDP

stabilized

Hachenberg

, 2001

Non-heart-

beating

livers

7,5000 U

SK in 20

mL Ringer

solution

Improved

structural

integrity

and

functional

& metabolic

recovery

Table Streptokinase studies on isolated organs

63

TIMELINE OF RESEARCH PROGRESSION

Constant Pressure: Test 1 – 2

Constant Flow: Test 3 – 13

Sterilized Plegisol: Test 1 – 5

Modified Plegisol: Test 5 – 13

Heparin: Test 1 – 6

Streptokinase: Test 6 – 13

UW (Transport Only): Test 2 – 7

Plegisol (Transport Only): 8 – 13

Insulin: Test 4 – 6

Epinephrine: Test 1-6, 9, 11, 13

Defibrillator and Pacing: Test 8 - 13

Catheterization and Pressure Transducer: Test 11 – 13

64

BLOOD CLOTS IN TEST 8 IN CHAMBERS

Figure 4.1.7 – Despite all washes performed, post-experiment

dissection showed heavy blood clots in cardiac chambers such as

near the chordae tendineae

65

TEST 11, N = 21

66

AORTIC PRESSURE FOR N = 22

Figure 4.1.13 – Aortic pressure mean data acquired for n = 22 67

TEST 11

Figure 4.1.15 – ventricular tachycardia for n = 21

68

TEST 11

Figure 4.1.17 – Mean aortic pressure for n = 21

69

TEST 13, N = 26 ECG

Figure 4.1.22 – (top) ventricular tachycardia leading to fibrillation

of n = 26. (Bottom) Stabilized heart rate n = 26 half an hour later

70

AORTIC PRESSURE

Figure 4.1.24 – aortic pressure for n = 25 stabilized at

t = 1 hour at 74.7 mmHg

71

RATE OF AORTIC PRESSURE FOR TEST 13

Figure 4.1.25 – pressure of continuous aortic pressure for n = 25, 26

0

20

40

60

80

100

120

140

3 7 17 19 66

Pre

ssu

re

(m

mH

g)

Time (Minutes)

Mean Aortic Pressure

n = 23

n = 24

72

TRANSMEDIC ORGAN CARE SYSTEM: HEART ™ [10]

Photo credit: http://www.transmedics.com/wt/page/organ_care

http://tesi.cab.unipd.it/36410/1/Tesi.pdf

Ex vivo resuscitation:

• build up its energy stores

• optimize its function

• perform full viability assessment

prior to transplantation

73