NutriChip, Nano-Tera annual meeting Bern 2013

7/28/2019 NutriChip, Nano-Tera annual meeting Bern 2013

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NutriChip

Annual Plenary Meeting, Bern, May 30th 2013


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NutriChip

Project

NutriChipGroup Gijs:

microfluidics

bioMEMS

cell-based & particlehandling in systems

Group Ramsden:

interfacial interactions in

aqueous systems

cellomics (cell-on-chip)

complex systems

modeling and design

Group Carrara:

Integrated Nano-Bio-systems

use of CMOS design and

technology for bio-sensing

purposes

Group HurrellHuman nutrition

strategies to combat

micronutrient deficiencies and

chronic diseases

theoretical aspects of

nutrition

Group Vergres:Biochemistry & Physiology

of dairy products

Human nutrition

Nutrigenomics

Biochemistry
http://www.microsens.ch/http://www.ayanda-biosys.com/index.html


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General objective

To develop a microfluidic analytical platformmimicking the gastro-intestinal tract for

screening the health-promoting properties ofmilk and dairy products.


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60000 kg

(40000 pills)

20 kg

Food more than a confounding factor

For each gram ingested, the number of medical publications is 30000

times higher for drugs than for food (G. Vergres).

. . at the same time, 30-40% of cancers are associated with nutrition.


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NutriChip: biological principle

MilkLPS

High-fat diet

High-calory diet

Epithelium

Immune cell

Cytokines

lipopolysaccharide (LPS)

Interleukins (IL),

Tumor Necrosis Factor-a (TNF-a), ..

Can milk act against

inflammation ?

Gastro-

intestinal

Tract (GIT)


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NutriChip: biological principle

MilkLPS

High-fat diet

High-calory diet

Epithelium

Immune cell

Cytokines ?

lipopolysaccharide (LPS)

Interleukins (ILs),

Tumor Necrosis Factor-a (TNF-a), ..

T. T. McDonald, Nature Medicine 16, 1194 (2010).


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NutriChip: a gut-on-a-chip

Gastrointestinal

Tract (GIT)

Epithelial Cells

(Caco-2)

Cytokines

detection

Differentiated

monocytes (U937)

Functionalized

magnetic beads

Epithelial cellsculturing &

differentiation

Monocytesculturing &

differentiation

Epithelial cells (Caco-2)

Immune cells

Immunomagnetic

assay


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NutriChip project

CMOS imagersystem

Transwell-

based GIT

Human study

Calcium bio-

availabality+ Ca-NutriChip

Nutrichip

(micro-GIT)


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CMOS camera


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United Microelectronics Corp.0.18 m process

Mini ASIC :

1525 m x 1525 m

Different photodiodes

Different pixel architectures

Different noise reduction circuits

Multiphase clock generator

CMOS first chip-prototype: test photodiodes


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Noise Reduction: Correlated Double Sampling perColumn

Column Scanning: Shift Registers

Row Scanning: 6 bit Counter

United

Microelectronics Corp.

0.18 m process

Mini ASIC:

3240 m x 1525 mResolution:

64 x 40 pixel array

Testing from July 2013

CMOS pixel array chip


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Synthetic image generation

Generation of random fluorophore cluster

using a Monte-Carlo approach

Cell population

For each cell: fluorophore clustersgeneration

Imaging simulation from the location of the

fluorophores

Simulation of the optical system

(convolution with the point spread function)

Simulation of the CCD/CMOS imager

(shadowing, noise, exposure time,

sampling)

Simulated data used to test and validate fluorescence image

processing methods


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Synthetic image processing

Thresholding Adaptive image thresholding

Fluorescent pixels

Average fluorescent

pixel intensity per cell

Number of fluorescent

pixels per cell

Quantifying and evaluating the amount of

fluorescent targets from epi-fluorescence

microscopy images = estimating the number

of fluorescent targets

Objective

~ measured number offluorescent targets


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Original experimental

image

Fluorescent image processing

Codes developed in Matlab and translated for CMOS-embedded

application.

Discretization of

fluorescent spots

Removal of low-varying

background by Top-HatFiltering

O ll l i f th


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Overall conclusions for the

CMOS imager system

n-well/p-sub type photodiodes are recommended for higher sensitivity

n+/p-sub type photodiodes have the smallest area but cannot be used

for low-intensity light imaging applications

Optimum fluorophores need to emit light at wavelengths from 630 nm

to 780 nm to reach the highest responsivityand sensitivityof the

CMOS detector.

Local thresholding method was proven to be the best for

classifying/localizing fluorescent biomarkers on CaCo-2 cells


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In vitrodigestion of dairy food

30 kDa

filtration

- Gel electrophoresis

- size-excl. High Performance Liquid Chromatography

- OPA labeling of di-and tripeptides, free amino acids

37 oC

Analysis of

digested product :

OPA: ortho-phtalaldehyde


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Pasteurized milk:

undigested and

digestedat different stages

Undigested Digested

5 min Saliva

120 min

gastric juice

30 minpancreatic

juice & bile


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Analysis by liquid chromatography-mass spectrometry (LC-MS) or MS

Peptide length distribution afterin vitro digestion of milk



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Transwell: Caco-2 differentiation and integrity

0

100200

300

400

500

600

700

800

900

0 5 10 15 20 25

Relativeamounts[%]

time [days]

Alk.P. and Lct expression in Caco-2

ap lct

Alkaline phosphatase (AP) activity

(signature of tight epithelial cell

junctions)

Lactase expression (signature of

Caco-2 differentiation) Trans-Epithelial Electrical

Resistance (TEER)

Permeability to FITC-dextran or

Lucifer Yellow

Caco-2 seeding in

Transwell

21 days 10% in FBS

culture medium

2 hours 0.2% serum

Treatment

with digested

products

Detection of

biomarkers

T ll C 2 l t


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Transwell: Caco-2 layer response to

stimulation

0

200

400

600

800

1000

1200

0 20 40 60 80 100 1 20

IL-6

[pg/ml]

TNF-a [ng/ml]

0

200

400

600

800

1000

1200

0 4000 8000 12000

IL-6

[pg

/m

l]

LPS [ng/ml]

Caco-2 layer is sensitive to

TNF-, but not to LPS, in

terms of basolateral IL-6induction

IL-6 is measured by ELISA

TNF-a apical

TNF-a basolateral

0

20

40

60

80

100

120

140

160180

UT apical basolateral both

IL-6

[pg/ml]

T ll lt t TNF d


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TNF-a, LPS apical

Transwell: co-culture response to TNF-a andLPS stimulation

0

1020

30

4050

6070

80

Baso

latera

lIL-6

[pg

/m

l]

LPS (1) 1 g/mL

LPS (10) 10 g/mLTNF-a applied at 10

ng/mL concentration


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Overall conclusions for the Transwell system

TNF-a induces a decrease of TEER in Caco-2

U937 immune cells (human leukemic monocyte lymphoma cell line)

treated with phorbol ester PMA express TNF-a

TNF-a induces IL-8 basolateral secretion in co-culture experiments

LPS induces IL-6 expression and secretion in U937 cells

The in vitro digested products do not markedly influence Caco-2 or

U937 cells and do not interfere with LPS or TNF-a application

Ca availability in dairy products investigated


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NutriChip design

Design of a miniaturized device for culturing Caco-2 cells with concurrent4-point measurement of the Trans-Epithelial Electrical Resistance (TEER)

4-p

ointTEER


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NutriChip realization

Fluidic inlet top layer

Fluidic inlet bottom layer Top electrode (current)

Bottom electrode (voltage)

Channel height: 250 m 20 m

Fluidic outlet bottom layerBottom electrode (current)

Fluidic outlet top layer

Top electrode (voltage)

1 mm


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Caco-2 cell layer characterization

Apparent permeability coefficient for FITC-labeled dextran:

Papp (cm/s) = P (mol/s) / (A (cm2) C0(mol/mL))

with

Pthe permeability rate, C0 the initial concentration in the upper chamber,

andA the surface area of the monolayer

Papp 710-4 cm/s


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4-point measurement of the Trans-Epithelial Electrical Resistance (TEER)


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Transwell

NutriChip


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On-chip immunomagnetic assay

Differential response of monocytic cells to LPS stimulation



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miniaturized GIT (NutriChip)

Microfluidic model of the gastrointestinal tract realized

Caco-2 culture performed

Layer integrity tested by FITC-dextran transport study

TEER results confirmed confluency of the cell monolayerobtained after 6 days of culture

Magnetic-bead based immunoassay developed

Ca-transport study through the cell monolayer in progress


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Meal A

Meal B

Meal C

37 persons

3 times @ Inselhospital, Bern to consume

meals A, B, and C

5 blood samplings per Person/Day/Test meal 570 blood sampling time points

Objective:

Evaluate the effect of a high-fat meal on postprandialmetabolism and inflammation in normal weight and

obese subjects

500kcal

1000 kcal

1500 kcal

Human nutrition study

18 19


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Meal A

Meal B

Meal C

Analysis: Classical clinical parameters: glucose, insulin,

triglycerides, total cholesterol, high-density

lipoprotein(HDL)-cholesterol

Inflammation mediators: C-reactive protein (high-sensitivity CRP), IL-6, endotoxin (lipopolysaccharide

LPS))

Antihyperglycemic hormone : glucagon-like peptide-1

(GLP-1)

Other technologies: transcriptomics, metabolomics

500kcal

1000 kcal

1500 kcal

Human nutrition study


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Postprandial glucose and insulin response

0

1

2

3

4

5

6

7

8

0 2 4 6

mmol/L

500kcal1000kcal

0

1

2

3

4

5

6

7

8

0 2 4 6

mmol/L 500 kcal

1000 kcal

1500 kcal

0

20

40

60

80

100

120

140

160

0 2 4 6

mU/L

500 kcal

1000 kcal

1500 kcal

time (h)

time (h)

time (h)

Glucose

Insulin

Glucose

time (h)

Insulin

+iAUC meal A

0

20

40

60

80

100

120

140

160

0 2 4 6

m

U/L 500 kcal

1000 kcal1500 kcal

+iAUC: positive

incremental area under the

curve

D f l d i li


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Dose-response for glucose and insulin

0

0.5

1

1.5

2

2.5

3

3.5

4

4.55

500 kcal 1000 kcal 1500 kcal

Glucose 0h - 6h

0

50

100

150

200

250

300

350

400

500 kcal 1000 kcal 1500 kcal

Insulin 0h 6h

Significant increase in +iAUC from meal A to meal B to meal C

(glucose only in obese, insulin for both groups) Significant difference between obese and normal weight in the dose-

response

+iAUC for meal C in normal weight still below +iAUC for meal A in

obese

meal Bmeal A meal C

+iAUC: positive incremental area under the curve

meal Bmeal A meal C

x

b

a

c z

y

a

ab

b

x

y

z

+iAUC(mU/L*h)

+iAUC(mmol/L*h)


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Overall conclusions for the human study

Methodological basis for nutrition intervention study onhuman subjects with a different metabolic status

Dose-dependent effect of a high-fat meal on metabolic

parameters demonstrated

Difference between normal weight and obese in the doseresponse (metabolic parameters, IL-6)

Triglycerides may play a role in endotoxin absorption

Obese had higher baseline levels of GLP-1, but responded

similar or less strong (meal C) to the different meals

compared to the normal weight


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March 15th 2012

Thanks for your attention!

The NutriChip team
http://www.sf.tv/


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Backup slides


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Quantification of individual free amino acids (AA) by HPLC

after Stage 3 digestion (Saliva, Gastric, Pancreatic juices and Bile)

Transwell: integrity of Caco-2 layer


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LPS, TNF-a apical

Transwell: integrity of Caco-2 layer

with TNF-a and LPS stimulation

020406080

100120140

160180

Re

lativeTEER

LPS (1) 1 g/mL

LPS (10) 10 g/mL

TNF-a applied at10 ng/mL

concentration

050100

150200250300350400450

Relative4

kDaFITC

permea

bility

P di l I fl i


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Postprandial Inflammation

A. N. Margioris, Current Opinion in Clinical Nutrition and Metabolic Care 2009, 12:129137

1 2 3 4 5 6 7 8

Postprandial time in hours

Inflammationand

oxid

ationmarkers

Meal

Maladaptation

dysmetabolism

Normal

Adaptation +2SD

Meals high in calories, carbohydrate and Saturated

fatty acidsproduce a strong postprandial

immune/inflammatory response;

Postprandial stress as an indicator of


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1 2 3 4 5 6 7 8

Postprandial time in hours

Inflammationand

oxidationmarkers

Meal

Maladaptation

dysmetabolism

Normal

Adaptation

+2SD

A. N. Margioris, Current Opinion in Clinical Nutrition and Metabolic Care 2009, 12:129137

Postprandial stress as an indicator of

metabolic health

Meals high in calories, carbohydrate and Saturated fatty acids produce

a strong postprandial immune/inflammatory response;

Characterization of milk products


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Characterization of milk products

Collection of data of 2-D gel electrophoresis and LC-MS identification in

an interactive database

Information about identified proteins (Uniprot)

Result tables and comparison between different

dairy products possible

From 15 selected dairy products ~ 2000 proteins wereidentified (450 were different proteins)

Each product has a unique proteome and might

produce a different inflammatory response

In vi tro digestion of milk


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In vitrodigestion of milk

Digestion of pasteurized milk

past.milk

saliva

pH 6.8

gastric

juicepH 2-3

pancreatic juice

pH 6.5-7

bile

pH 6.5-7

5 min 2 h 2 h

Application

on the cell

culture

system

(modified from Versantvoort et al., 2005)

Model Versantvoort: used for the detection of bioavailability of

mycotoxins

Aim in our study: detect effect on inflammation

+ Analysis of

macro-nutrient

digestion


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N t iChi i l ti f h t


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Media flow velocity should be less

than 0.1 m/s for safe shear stress on

cells

Contours of wall shear

stress (Pascal)

Contours of velocity

magnitude (m/s)

Flow

NutriChip: simulation of shear stress

N triChip microfabrication aspects


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A procedure for irreversiblebonding of the porous membrane

in between two PDMS layers was

developed

Cell culture -chamber

with perfusion channels

NutriChip: microfabrication aspects

Cells on the NutriChip


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Epithelial cells (Caco-2) and monocytes (U937) cells are being separately

cultured in the microfluidic chips

Caco-2 U937

Cells on the NutriChip

Ca-NutriChip


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Ca-NutriChipDigestion of Emmental cheese with/without CaCl2 in the digestion juices

Emmental Cheese(1,2,3 and 4g)

digested with/without CaCl2

Amount of free amino acidsAmount of free fatty acids

Ca-NutriChip


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Ca-NutriChipDigestion of different dairy products to determine Ca2+ amount in the digests

5min 2h 2h

200 L saliva

pH 6.8

400 L gastric juice

pH 2-3

400 L pancreatic

juice pH 6.5-7

200 L bile

pH 6.5-7

&

+ +

Saliva contains 2 mmol/L ofCa2+

Gastric juice contains 0.6 mmol/L ofCa2+

Pancreatic juice contians 0.6 mmol/L ofCa2+

Bile contains 3.7 mmol/L ofCa2+

CaCl2 OMITTED from the digestion!

Filtration of the

digest

through 30

kDa


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Ca-NutriChip: NutriChip for study of


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Digestion of different cheeses & determination of the amount of Ca

in the digests by a colorimetric assay

Gruyre

Emmentaler

Tilsiter

Sbrinz

Vacherin fribourgeois

Appenzeller

3 mg of

protein

digested

without CaCl2

in digestion

juices

Semi-hard and hard cheeses are rich sources of calcium

Ca NutriChip: NutriChip for study of

nutrikinetics

Ca-NutriChip


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Amount of free amino acids

mMo

ffreeaminoacids

Ca-NutriChipDigestion with/without CaCl2 in the digestion juices

The efficency of digestion is not influenced by omitting

calcium chloride from the digestion juices

Fat & proteins in milk, yogurt and cheese are well digested.

Ca-NutriChip


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Ca NutriChip

Stimuli: various dairy

products

Ca-NutriChip

Epithelial Cells

(Caco-2)

Ca2+Ca2+ Ca2+ Ca2+

Transported Ca2+

through Caco-2 cells

Ca2+ uptake by target

cells

THP-1 cells

Osteoblast-like cells

Magnetic beads

Ca2+Ca2+ Ca2+ Ca2+

Target cells (Ca2+ sink)

Variety of dairy

product-based Ca

concentrations.

Extending the functionality of Nutrichip with a nutrikinetic capability

Ca-NutriChip: Nutrichip for study


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No Ca2+

No FRET

+ Ca2+

CFP & YFP are close

FRET

440 nm

480 nm 530 nm

Ratio

Intracellular Ca2+ imaging with Frster resonance energy transfer (FRET)

Ca NutriChip: Nutrichip for study

of nutrikinetics

Publications and media coverage


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Publications and media coverage

Journals4 submissions

Conferences25 oral and poster presentations

Media coverage
http://www.sf.tv/http://www.cdrf.org/

NutriChip, Nano-Tera annual meeting Bern 2013

Documents

Transcript of NutriChip, Nano-Tera annual meeting Bern 2013