Lecture #21: Case Study€¦ · • As liver function deteriorates, one or more complications may develop. In some people, complications may be the first signs of the disease. •

Lecture #21: Case Study

Liver Cirrhosis

Liver Cirrhosis

• In the United States, heavy alcohol consumption and chronic hepatitis C have been the most common causes of cirrhosis.

• Obesity is becoming a common cause of cirrhosis, either as the sole cause or in combination with alcohol, hepatitis C, or both.

• Many people with cirrhosis have more than one cause of liver damage.

Liver Cirrhosis

Nonalcoholic fatty liver disease (NAFLD)Nonalcoholic steatohepatisis (NASH)

Starley et al., Hepatology, 51, 1820, 2010

What are the complications of cirrhosis?

• As liver function deteriorates, one or more complications may develop. In some people, complications may be the first signs of the disease.

• Bruising and bleeding. When the liver slows or stops producing the proteins needed for blood clotting, a person will bruise or bleed easily.

• Esophageal varices and gastropathy. When portal hypertension occurs, it may cause enlarged blood vessels in the esophagus, called varices, or in the stomach, called gastropathy, or both. Enlarged blood vessels are more likely to burst due to thin walls and increased pressure. If they burst, serious bleeding can occur in the esophagus or upper stomach, requiring immediate medical attention.

• Splenomegaly. When portal hypertension occurs, the spleen frequently enlarges and holds white blood cells and platelets, reducing the numbers of these cells in the blood. A low platelet count may be the first evidence that a person has developed cirrhosis.

What are the complications of cirrhosis?

• Jaundice. Jaundice occurs when the diseased liver does not remove enough bilirubin from the blood, causing yellowing of the skin and whites of the eyes and darkening of the urine. Bilirubin is the pigment that gives bile its reddish-yellow color.

• Gallstones. If cirrhosis prevents bile from flowing freely to and from the gallbladder, the bile hardens as gallstones.

• Hepatic encephalopathy. A failing liver cannot remove toxins from the blood, and they eventually accumulate in the brain. The buildup of toxins in the brain—called hepatic encephalopathy—can decrease mental function and cause coma. Signs of decreased mental function include confusion, personality changes, memory loss, trouble concentrating, and a change in sleep habits.

• Insulin resistance and type 2 diabetes. Cirrhosis causes resistance to insulin—a hormone produced by the pancreas that causes the body to absorb glucose from the blood. With insulin resistance, the body’s muscle, fat, and liver cells do not utilize insulin properly. The pancreas tries to keep up with the demand for insulin by producing more, but excess glucose builds up in the bloodstream causing type 2 diabetes.

Diagnosis

• Tests and procedures used to diagnose cirrhosis include:– Blood tests such as a complete blood count, bilirubin

test and specific tests to determine the cause of cirrhosis

– Imaging procedures such as computed tomography (CT), ultrasound and magnetic resonance imaging (MRI)

– Examination of a sample of liver tissue (liver biopsy), which is typically done using a needle guided by ultrasound imaging

PathogenesisHepatocellular carcinoma: cancer of the liverChronic Hepatitis

Treatments*

• Treatments for the underlying cause of cirrhosisIn early cirrhosis, it may be possible to minimize damage to the liver by treating the underlying cause. For example:– Treatment for alcohol dependency. People with

cirrhosis caused by alcohol use need to stop drinking. If stopping alcohol use is difficult, your doctor may recommend a treatment program for alcohol addiction.

– Medications to control hepatitis. Medications may control damage to liver cells caused by hepatitis B or C.

*Mayo Clinic

Treatments*• Treatments for complications of cirrhosis

– Excess fluid in your body. Fluid that accumulates in your abdomen (ascites) or your legs (edema) may be managed with a low-sodium diet and water pills. More severe fluid buildup may require procedures to drain the fluid or surgery to relieve pressure.

– Increased pressure in the portal vein and surrounding small veins. Blood pressure medications may control increasing pressure in the veins around your liver. This may prevent severe bleeding. Surgery to place a stent to hold open the portal vein also may be necessary. Your doctor may recommend an endoscopy procedure to examine the veins in your esophagus and stomach for signs of bleeding.

– Infections. You may receive antibiotics or other treatments for infections.– Liver cancer screening. Your doctor may recommend periodic blood tests and

ultrasound exams to look for signs of liver cancer.– High levels of toxins in the blood (hepatic encephalopathy).Your doctor may instruct

you to watch for signs and symptoms of hepatic encephalopathy, which can range from confusion and mild changes in your thinking to coma. Medications can help treat hepatic encephalopathy.

– Liver transplant surgeryPeople with advanced cirrhosis may require liver transplants if their livers are no longer functioning (liver failure). Liver transplant is a procedure to remove your liver and replace it with a whole liver from a deceased donor or with part of a liver from a living donor.*Mayo Clinic

How Can We Treat These Patients?• Liver donation/transplantation

Liver transplant waiting list activity among adult patients

Liver transplant waiting list status, new listings (2006)

United Network for Organ SharingOrgan Procurement and Transplantation Network (2010)

22.8%

Cadaveric transplantation• The first human liver transplantation was in 1963• Until the application of cyclosporine for

immunosuppression in the late 1970s, the long-term survival of transplant recipients was low.

• Because of continued improvements in surgical techniques, organ preservation, and immunosuppression, 1-year survival rates are currently 85–90%.

• The most common indications for liver transplantation are chronic hepatitis, alcoholic liver disease, and cirrhosis. – The standard immunosuppressive therapy for transplant

recipients consists of tacrolimus plus corticosteroids.

Split liver transplantation

• In efforts to more effectively distribute donated organs, surgeons have developed techniques to use a single liver in two recipients.

• Split liver procedures originated with reduced-size transplantation, in which a child would receive only the right lobe of a cadaveric liver. – It soon became evident that the larger left lobe could be

successfully grafted into adults, rather than discarded.• Split liver operations are not performed everywhere

and may be complicated by anatomical variation or the availability of recipients meeting specified criteria.

Xenotransplantation

• The earliest reports of hepatic xenograftscame in 1993, with baboon liver transplantation being utilized

• FDA has approved trials to continue– Risks?

Xenotransplantation

• The earliest reports of hepatic xenograftscame in 1993, with baboon liver transplantation being utilized

• FDA has approved trials to continue– Risks?

• Rejection• Infectious risk

What are our Options?

• Tissue Engineering has provided us with the tools needed to attempt to solve the problem of liver cirrhosis

What are the Challenges?(The 3 S’s)

• Sourcing (cell source)Primary donor cells, stem cells, xenogeneic



• Scale (must achieve clinically relevant scale)Cell sourcing, delivery and engraftment, transport




• Stability (maintenance of cell function)Cell-microenvironment, biomaterial, 3D structure

In-Class Exercise

Cells Used in Extracorporeal Solutions

• Primary hepatocytes are the most common cellular component in current engineered therapies.

• Most devices undergoing clinical evaluation use porcine hepatocytes, which are readily available.

Dhawan, A. et al. (2010) Nat. Rev. Gastroenterol. Hepatol.

Delivery & Engraftment

Miller et al., Nature Materials, 2012 Homework #2

Transport Limitations

Extracorporeal Devices• The most common bioartificial liver device design

incorporates hepatocytes in hollow fiber cartridges borrowed from hemodialysis.

• Hollow fiber membranes provide a scaffold for cell attachment and immunoisolation, and are well characterized in a clinical setting, but may not provide adequate nutrient transport or the proper environmental cues for long-term hepatocytestabilization.

• Flat plate or monolayer bioreactors have been proposed that offer better control of hepatocytemicroenvironment, but would be difficult to scale up.

Extracorporeal Devices

Extracorporeal Device Needs• Although many devices include a combination of convective and diffusion

transport phenomena, mass transfer limitations of key nutrients to and from the cellular compartment are primarily due to diffusion resistance.

– Barriers to diffusive transport include membranes, collagen gels, and nonviable cells. – Membranes have been used with a wide range of molecular mass cutoffs, from 20 to

200 kDa, but presently most designs specify a range between 100 and 150 kDa.

• Some challenges involved in scaling up experimental reactors include maintaining a uniform microenvironment, minimizing dead volume, providing adequate cell number, and ensuring homogeneous flow distribution.




• Stability (maintenance of cell function)Cell-microenvironment, biomaterial, 3D structure

Single-gel

Sandwich

35

0

1

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7 9 10 12 14

Day

Albu

min

(µg/

106

cells

/hou

r)

Effects of Co-Culture

Freshly Isolated Hepatocytes

Co-Cultured Hepatocytes 2 weeks later

In vivo levels

Chart2 (2)

10.22311416390.02877009940.02877009940.02490986380.0249098638

20.40858519490.01482588750.01482588750.08792570520.0879257052

30.11562323750.01382456290.01382456290.00858564690.0085856469

40.93516273130.00754184130.00754184130.0552734830.055273483

51.34832786890.00015143610.00015143610.21442280710.2144228071

65.6505424378000.43700450480.4370045048

76.85351724140.00128159630.00128159630.18511461670.1851146167

97.09969892470.00012903230.00012903230.1281480240.128148024

108.0357142857000.25159111810.2515911181

127.93852255830.00009915720.00009915720.05440900960.0544090096

148.479257732000.30249590710.3024959071

Pure Hepatocytes

Hepatocytes with J2-3T3s

0.2813754293

0.1417433202

0.0562887761

0.0195277601

0.0002622951

0

0.0053793103

0.0001290323

0

0.0000991572

0

Chart1

10.02788927050.02510655030.02284106430.02085488480.00359626240.0031137330.00180273760.00189093750.0014816632

20.05107314940.02329172140.02274419620.01286684190.00185323590.01099071320.00386321090.00248499240.0018904663

30.01445290470.0113931190.0092498590.00169204740.00172807040.00107320590.00127143560.00160521710.0001151292

40.11689534140.10406828330.07037332480.00023931080.00094273020.00690918540.01645504340.01920910970.0000305497

50.16854098360.5215573770.407081967200.00001892950.02680285090.0661886330.05239689630

60.70631780470.5655392470.3446553925000.05462556310.0323807160.02685644560

70.85668965520.89708620690.56843103450.00031034480.00016019950.02313932710.04797209330.03730670490.000044517

81.32358730161.15773015871.0559365079000.07044307580.09300559160.12703184820

90.88746236560.8564838710.434951612900.0000161290.0160185030.09910978190.02118387160

101.00446428570.70177176340.3633510045000.03144888980.11183722650.02787069860

111.51676570460.7258771930.24107243920.00052348610.00007121140.03807931370.01422972920.02197424930.0001092872

120.99231531980.81357213680.32878036690.00001239460.00001239460.00680112620.10130206060.05174968010.0000123946

141.05990721650.59617525770.237185567000.03781198840.04866067080.02127986970

MEF/HC coculture

J2/HC

NIH/HC

L1/HC

HC

days of co-culture

albumin (microgram / mL / hour)

Albumin secretion (co-culture of hepatocytes with 3T3 fibroblasts, exp 4-10-02)

0.0351719287

0.017717915

0.007036097

0.00244097

0.0000327869

0

0.0006724138

0

0.000016129

0

0.0003678551

0.0000123946

0

Chart2

110.02490986380.02490986380.02877009940.0287700994

220.08792570520.08792570520.01482588750.0148258875

330.00858564690.00858564690.01382456290.0138245629

440.0552734830.0552734830.00754184130.0075418413

550.21442280710.21442280710.00015143610.0001514361

660.43700450480.437004504800

770.18511461670.18511461670.00128159630.0012815963

990.1281480240.1281480240.00012903230.0001290323

10100.25159111810.251591118100

12120.05440900960.05440900960.00009915720.0000991572

14140.30249590710.302495907100

Hepatocytes with J2-3T3

Pure Hepatocytes

0.2231141639

0.2813754293

0.4085851949

0.1417433202

0.1156232375

0.0562887761

0.9351627313

0.0195277601

1.3483278689

0.0002622951

5.6505424378

0

6.8535172414

0.0053793103

7.0996989247

0.0001290323

8.0357142857

0

7.9385225583

0.0000991572

8.479257732

0

analysis

DayHours1 avg1 stderr2 avg2 stderr3 avg3 stderr4 avg4 stderr5 avg5 stderr

124.1670.850.040.090.000.670.030.080.000.610.030.040.000.500.020.040.000.550.020.050.00

222.830.400.020.040.001.170.050.250.010.530.020.090.000.290.010.040.000.520.020.060.00

317.730.120.010.030.000.260.010.020.000.200.010.020.000.030.000.000.000.160.010.030.00

415.670.040.000.010.001.830.120.110.011.630.100.260.020.000.000.000.001.100.070.300.02

515.250.000.000.000.002.570.170.410.037.950.521.010.070.000.000.000.006.210.410.800.05

615.670.000.000.000.0011.070.710.860.058.860.570.510.030.000.000.000.005.400.340.420.03

714.50.010.000.000.0012.420.860.340.0213.010.900.700.050.000.000.000.008.240.570.540.04

815.750.000.000.000.0020.851.321.110.0718.231.161.460.090.000.000.000.0016.631.062.000.13

915.50.000.000.000.0013.760.890.250.0213.280.861.540.100.000.000.000.006.740.430.330.02

1017.920.000.000.000.0018.001.000.560.0312.580.702.000.110.000.000.000.006.510.360.500.03

1117.670.010.000.000.0026.801.520.670.0412.830.730.250.010.010.000.000.004.260.240.390.02

1220.170.000.000.000.0020.020.990.140.0116.410.812.040.100.000.000.000.006.630.331.040.05

1448.50.000.000.000.0051.411.061.830.0428.910.602.360.050.000.000.000.0011.500.241.030.02

avg hours20.10

stdev hours9.04

DayJ2/hepHep8

10.220.020.2810.03

20.410.090.1420.01

30.120.010.0560.01

40.940.060.0200.01

51.350.210.0000.00

65.650.440.0000.00

76.850.190.0050.00

810.590.560.0000.00

97.100.130.0000.00

108.040.250.0000.00

1112.130.300.0030.00

127.940.050.0000.00

148.480.300.0000.00

plate1

SampleConcentrationBack Calc ConcWellsOD ValuesMean OD ValueStd.Dev.CV%SampleWellsOD ValuesConcentrationMean Conc.Std.Dev.CV%DilutionAdj. Conc.AvgStdErr

St0110073.685A10.0580.0590.0022.9Un01A40.3190.5951.090.51747.411.090.8500.087

70.743A20.061A50.2641.626

75.341A30.057A60.2881.048

St025054.382B10.0750.0770.0033.3Un02B40.3730.2170.7250.5077010.725

53.682B20.076B50.2791.232

49.848B30.08B60.3080.726

St032528.229C10.110.1080.0022.1Un03C40.3160.630.8660.20924.210.866

28.757C20.109C50.2891.029

30.569C30.106C60.2940.938

St0412.512.108D10.1560.1520.0085.5Un04D40.3420.3880.7190.32445.110.719

15.606D20.142D50.2891.037

11.798D30.157D60.3070.732

St056.256.136E10.1930.1720.02212.7Un05E40.330.480.8960.48153.710.8960.6740.075

8.659E20.174E50.3040.785

13.734E30.149E60.2721.423

St063.1252.686F10.2370.2220.0146.4Un06F40.3280.50.5620.0549.610.562

3.791F20.219F50.3180.6

4.52F30.209F60.320.585

St071.5630.585G10.320.290.0269Un07G40.3430.3760.6250.22235.510.625

1.196G20.281G50.3020.803

1.46G30.27G60.310.696

St0800H11.1031.0420.0656.2Un08H40.3330.4570.6130.18630.410.613

0H21.049H50.3220.563

0H30.973H60.3010.819

Un09A70.3070.7440.6580.1322010.6580.6070.044

r20.966A80.3270.506

A90.3080.722

Un10B70.3380.4180.5350.1552910.535

B80.3310.476

B90.3090.71

Un11C70.2990.8610.7030.20328.810.703

C80.3310.474

C90.3050.772

Un12D70.3250.5310.5310.0010.110.531

D80.3250.53

D90.3250.531

Un13E70.3210.5670.5430.06111.310.5430.5040.036

E80.3190.588

E90.3310.473

Un14F70.3550.3050.4060.09222.610.406

F80.3360.428

F90.330.484

Un15G70.3240.5430.5690.0579.910.569

G80.3250.529

G90.3150.633

Un16H70.3160.6250.4980.19238.610.498

H80.360.277

H90.3190.593

Un17A100.3040.7840.6830.152210.6830.5520.046

A110.3060.754

A120.3270.51

Un18B100.320.5820.4990.07514.910.499

B110.3350.436

B120.330.48

Un19C100.3150.6360.5450.0821510.545

C110.3310.476

C120.3260.523

Un20D100.3510.3270.4810.22446.510.481

D110.3430.378

D120.3070.737

Un21E101.33700048.810

E111.370

E121.3870

Un22F101.4040003910

F111.4050

F121.4470

Un23G101.38400035.210

G111.4220

G121.4060

Un24H101.4230003.510

H111.4220

H121.4260

Sheet2


St0110075.039A10.070.0720.0011.4Un01A40.4020.2890.3720.09826.510.3720.4050.042

72.563A20.072A50.3910.346

73.42A30.072A60.3710.481

St025052.756B10.0910.0950.0055.1Un02B40.3980.3080.5310.24345.810.531

44.983B20.101B50.3420.79

50.42B30.094B60.370.496

St032532.595C10.120.1190.0032.4Un03C40.3680.510.3560.19354.210.356

31.678C20.122C50.380.418

34.799C30.116C60.4450.14

St056.258.502E10.20.1880.0126.5Un04D40.4280.1850.3590.15142.110.359

10.242E20.189D50.3740.457

12.781E30.176D60.3780.434

St063.1252.588F10.2710.2730.0114.1Un05E40.3261.0281.0080.58357.811.0081.1660.251

2.999F20.262E50.3011.58

2.063F30.285E60.380.415

St071.5630.859G10.3370.3160.0185.8Un06F40.4040.2781.2810.88368.911.281

1.536G20.302F50.2881.942

1.375G30.309F60.2991.623

St0800H11.1811.1670.0121Un07G40.2961.7131.7860.38921.811.786

0H21.161G50.3061.438

0H31.16G60.2812.206

Un08H40.3780.4310.5890.44174.810.589

H50.3231.087

H60.4110.249

Un09A70.3550.6320.6150.0243.810.6150.5320.088

A80.3560.625

r20.962A90.3590.588

Un10B70.3141.2680.7120.60785.310.712

B80.3410.804

B90.4910.064

Un11C70.3340.9050.4980.40982.210.498

C80.4740.087

C90.3690.502

Un12D70.4920.0640.3020.375124.410.302

D80.4610.107

D90.3460.734

Un13E70.4850.0710.2220.271122.310.2220.2940.043

E80.4960.06

E90.3650.535

Un14F70.370.4960.380.16443.210.38

F80.4260.192

F90.3750.452

Un15G70.3620.5650.3560.1935410.356

G80.4280.185

G90.3960.319

Un16H70.4380.1580.2170.08338.510.217

H80.3970.312

H90.430.18

Un17A100.3630.5530.4540.1839.710.4540.5190.057

A110.3620.562

A120.4110.246

Un18B100.3630.5520.5580.0254.510.558

B110.360.586

B120.3650.537

Un19C100.430.1810.4050.422104.310.405

C110.3350.892

C120.4440.142

Un20D100.3640.5480.660.27341.410.66

D110.330.971

D120.3740.46

Un21E101.3890009610

E111.4440

E121.5460

Un22F101.41800070.510

F111.4690

F121.5020

Un23G101.4600053.410

G111.4470

G121.5230

Un24H101.4810003310

H111.5210

H121.50

Sheet3


St0110075.527A10.0640.0630.0011.2Un01A40.4320.0920.1410.05841.510.1410.1250.031

77.056A20.062A50.3880.205

77.619A30.062A60.4150.125

St025048.875B10.0870.0830.0044.9Un02B40.3960.1780.20.05226.110.2

56.54B20.079B50.4010.163

53.632B30.082B60.3750.26

St032528.199C10.1180.1130.0054.1Un03C40.4870.0340.1030.0747210.103

33.282C20.109C50.3950.181

31.113C30.112C60.4320.093

St0412.512.07D10.1640.1540.0117.4Un04D40.4830.0370.0550.02443.910.055

18.149D20.142D50.4380.082

13.861D30.157D60.470.046

St056.257.49E10.190.1910.0073.5Un05E40.3710.2810.2850.08429.410.2850.2560.019

8.416E20.184E50.3560.37

6.606E30.197E60.3890.203

St063.1252F10.2630.2560.0062.2Un06F40.4220.1110.2040.214104.610.204

2.4F20.253F50.3450.449

2.395F30.253F60.4620.053

St0800H11.1111.0510.0565.3Un07G40.3990.1670.2520.16364.510.252

0H21.043G50.4050.15

0H31G60.3460.44

Un08H40.4130.1310.2840.13948.910.284

H50.3510.402

H60.3640.317

Un09A70.3740.2630.2020.0629.510.2020.2020.023

r20.968A80.4080.143

A90.3890.201

Un10B70.3610.3370.2250.09743.110.225

B80.3980.17

B90.3990.168

Un11C70.3760.2550.1390.1148210.139

C80.50.027

C90.4110.135

Un12D70.3520.3920.2420.13856.910.242

D80.4170.12

D90.3860.214

Un13E70.4630.0520.030.01962.710.030.0300.002

E80.5120.021

E90.5240.017

Un14F70.5040.0250.0350.0129.910.035

F80.4710.046

F90.4850.035

Un15G70.4620.0540.030.02480.510.03

G80.4960.029

G90.580.006

Un16H70.4950.0290.0250.01144.110.025

H80.4890.032

H90.5430.012

Un17A100.4170.1220.1620.1167.410.1620.1640.028

A110.440.079

A120.370.287

Un18B100.4530.0630.090.0273010.09

B110.4330.091

B120.4190.117

Un19C100.3840.2190.1760.0422.810.176

C110.3980.171

C120.4090.139

Un20D100.3640.320.2280.09742.810.228

D110.380.236

D120.4150.126

Un21E101.3710005110

E111.4030

E121.4270

Un22F101.35200098.310

F111.410

F121.4880

Un23G101.36900057.710

G111.3770

G121.4470

Un24H101.341000100.810

H111.4160

H121.450

Sheet4


St0110065.029A10.0750.0660.00811.4Un01A40.6540.0140.0250.0139.210.0250.0380.015

80.623A20.06A50.5990.031

75.932A30.064A60.60.03

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Control of Cell-Cell InteractionsH

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Slide Number 1Liver CirrhosisLiver CirrhosisLiver CirrhosisNonalcoholic fatty liver disease (NAFLD)�Nonalcoholic steatohepatisis (NASH)Nonalcoholic fatty liver disease (NAFLD)�Nonalcoholic steatohepatisis (NASH)What are the complications of cirrhosis?What are the complications of cirrhosis?DiagnosisPathogenesisTreatments*Treatments*How Can We Treat These Patients?Cadaveric transplantationSplit liver transplantationXenotransplantationXenotransplantationWhat are our Options?What are the Challenges?�(The 3 S’s)What are the Challenges?�(The 3 S’s)What are the Challenges?�(The 3 S’s)In-Class ExerciseWhat are the Challenges?�(The 3 S’s)What are the Challenges?�(The 3 S’s)Cells Used in Extracorporeal SolutionsWhat are the Challenges?�(The 3 S’s)What are the Challenges?�(The 3 S’s)Slide Number 28Slide Number 29Extracorporeal DevicesExtracorporeal DevicesExtracorporeal Device NeedsWhat are the Challenges?�(The 3 S’s)What are the Challenges?�(The 3 S’s)Slide Number 35Effects of Co-Culture �Slide Number 37

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Lecture #21: Case Study€¦ · • As liver function deteriorates, one or more complications may develop. In some people, complications may be the first signs of the disease. •