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Biomedical
Applications of
Nanotechnology
What is Nanotechnology?
The creation of useful, functional materials,
devices, and systems through:

1. Controlling and manipulating matter on the
   nanometer-length scale (1-100 nm), and
2. Exploiting novel phenomena and properties
   (physical, chemical, biological, mechanical,
   electrical) at the nanoscale.


ā€œGoing Small for Big Advancesā€
ā€œGoing Small for Big Advancesā€
Potential market for nanotechnology ?
$1 trillion business within the next 10 to 15 years.
Nanotechnology in Medicine

ā€¢ Biological imaging for medical diagnostics.
ā€¢ Advanced drug delivery systems.
ā€¢ Biosensors for airborne chemicals or other
  toxins.
ā€¢ Regenerative medicine:
  More durable, rejection-resistant
  artificial tissues and organs.

          NANOMEDICINE
          NANOMEDICINE
Targeted Drug Delivery




J Leukoc Biol 2005;78:585
Cardiovascular         Obesity /
                  Disease             Diabetes




Risk Factors               Inflammation              Nanotechnology



                                         Tumor
                Neurological
                                     Angiogenesis
                 Disorder
                                      / Metastasis
Development of
 In Vivo and In Vitro
Inflammatory Disease
        Models
In Vivo Brain Inflammation Models

ā€¢ Environmental Toxicants-Mediated
  Brain Inflammation.


ā€¢ Bacterial Infection-Mediated Brain
  Inflammation.
Environmental Toxicants & Brain Inflammation

Isolation of brain regions




                               C57/BL6 Mouse

 Determination of pro-inflammatory mediators
- Cytokines: TNF-Ī±, IL-1Ī², IL-6
- Chemokine: MCP-1
                                                    Chlorpyrifos
- Adhesion molecules: E-selectin, ICAM-1, VCAM-1
                                                   [OP Pesticide]
20                             *
                                  Control
(TNF-Ī± mRNA/GAPDH mRNA)
                                  CPF, 8 h
                                  CPF, 16 h
   Relative Fold Induction




                             15   CPF, 24 h
                                               *

                             10
                                                    *

                              5       *                         *
                                          *                          *
                                                        *                 *
                                  *
                              0
                                  HIP         COR       STR         CER
25
(TNF-Ī± mRNA/GAPDH mRNA)           Control
                                  CPF, 20 mg/kg              *
                                  CPF, 70 mg/kg
                             20
   Relative Fold Induction



                                  CPF, 140 mg/kg


                             15


                             10
                                        *          *    *
                              5    *                                   *
                                                                  *

                              0
                                  HIP         COR      STR       CER
6
                                 Control                                 *
                                 CPF, 4 h
(IL-6 mRNA/GAPDH mRNA)
                             5   CPF, 8 h
   Relative Fold Induction


                                 CPF, 24 h

                             4                       *
                                       *

                             3                   *                  *
                                  *
                             2                                 *
                                             *

                             1

                             0
                                 HIP         COR         STR       CER
7
                                 Control
(IL-6 mRNA/GAPDH mRNA)
                                 CPF, 20 mg/kg
                                                             *
                             6   CPF, 70 mg/kg
   Relative Fold Induction


                                 CPF, 140 mg/kg
                             5

                             4    *                    *

                             3                                    *
                                                  *
                             2

                             1

                             0
                                 HIP         COR      STR   CER
6                     *
                                 Control
(MCP-1 mRNA/GAPDH mRNA)
                                 CPF, 8 h
                             5   CPF, 16 h
   Relative Fold Induction



                                 CPF, 24 h

                             4                                   *
                                      *

                             3                         *     *
                                             *
                             2

                             1

                             0
                                  HIP            STR       CER
5
(MCP-1 mRNA/GAPDH mRNA)          Control                      *
                                 CPF, 20 mg/kg          *
                                 CPF, 70 mg/kg
                             4
   Relative Fold Induction


                                 CPF, 140 mg/kg


                             3
                                         *
                                                  *
                             2


                             1


                             0
                                  HIP             STR       CER
3.0     *
(E-selectin mRNA/GAPDH mRNA)
                                                        Control
                                             *          CPF, 70 mg/kg
                                2.5
      Relative Fold Induction




                                                    *
                                2.0

                                1.5

                                1.0

                                0.5

                                0.0
                                      HIP   COR   STR     CER
Bacterial Infection & Brain Inflammation

Isolation of brain regions




                               C57/BL6 Mouse
 Determination of pro-inflammatory mediators
- Cytokines: TNF-Ī±, IL-1Ī², IL-6
- Chemokine: MCP-1
                                                   Lipopolysaccharide
- Adhesion molecules: E-selectin, ICAM-1, VCAM-1
                                                         [LPS]
40                                      30
                                      *                  Control
(TNF-Ī± mRNA/GAPDH mRNA)




                               35
                                                         LPS                  *
                                                                       25
     Relative Fold Induction




                                                                                                    *
                               30                    *                               *
                                                                       20
                               25
                                                                   *
                               20                                      15

                               15
                                                                       10
                               10
                                                                       5
                               5
                               0                                       0
                                    HIP      COR   STR        CER           HIP    COR   STR      CER



                                          4 h Exposure                            24 h Exposure
35                                12
(IL-6 mRNA/GAPDH mRNA)




                             30
                                   Control                 *                 *
                                   LPS
   Relative Fold Induction




                             25                                9

                             20
                                    *                          6
                             15               *
                             10
                                                     *         3                            *
                             5

                             0                                 0
                                  HIP        COR   STR   CER        HIP    COR   STR      CER


                                        4 h Exposure                      24 h Exposure
25                                      6
                                    *                  Control             *
(IL-1Ī² mRNA/GAPDH mRNA)




                                                       LPS
                                                                     5
                             20
   Relative Fold Induction




                                                                     4
                                                                 *
                             15
                                                                     3           *               *
                             10              *                       2
                                                   *
                             5
                                                                     1

                             0                                       0
                                  HIP      COR   STR         CER         HIP    COR   STR      CER


                                        4 h Exposure                           24 h Exposure
120                                       20
                                                         Control
(MCP-1 mRNA/GAPDH mRNA)




                                                         LPS
                                     *                                                              *
                             100
   Relative Fold Induction




                                              *                        15
                             80                                    *
                                                                                            *
                             60                                        10

                             40                      *                               *
                                                                       5
                             20

                              0                                        0
                                   HIP      COR    STR       CER            HIP    COR    STR     CER


                                         4 h Exposure                             24 h Exposure
15                   Control
                                                                   10
(ICAM-1 mRNA/GAPDH mRNA)




                                     *             LPS
                                                                          *
                                              *
    Relative Fold Induction




                              12                               *   8


                              9                                    6            *               *

                              6                      *             4
                                                                                       *
                              3                                    2


                              0                                    0
                                   HIP      COR   STR        CER        HIP    COR   STR      CER


                                         4 h Exposure                         24 h Exposure
20                                  15
(E-selectin mRNA/GAPDH mRNA)




                                      Control
                                      LPS
                                                                *                       *
      Relative Fold Induction




                                                                    12
                                15                                         *      *
                                                                    9                            *
                                10
                                                 *        *         6

                                5
                                       *
                                                                    3


                                0                                   0
                                     HIP        COR   STR     CER        HIP    COR   STR      CER

                                           4 h Exposure                        24 h Exposure
In Vitro Brain Inflammation Models

ā€¢ Brain
  Microvascular
  Endothelial Cells
  (BMEC)

ā€¢ Astrocytes

ā€¢ Microglia
Pro-inflammatory Stimuli-Mediated
           Inflammation in Brain Cells

Pro-inflammatory Stimuli
   [100 ng/ml of LPS]


                                                       Culture Media



                                                     Brain Cells

           Determination of pro-inflammatory mediators
          - Cytokines: TNF-Ī±, IL-1Ī², IL-6
          - Chemokine: MCP-1
          - Adhesion molecules: E-selectin, ICAM-1
Brain Microvascular Endothelial Cells (bEnd.3)



                             120                                                                          10
                                                       *
(TNF-Ī± mRNA/GAPDH mRNA)




                                                                             (IL-1Ī² mRNA/GAPDH mRNA)
                             100                                                                                                              *
                                                                                                          8
   Relative Fold Induction




                                                                                Relative Fold Induction
                             80
                                                                                                          6
                             60                                                                                                                      *
                                                                                                          4
                             40

                                                                  *                                       2
                             20
                                                                         *
                                             *
                              0                                                                           0
                                   Control   1         2          4      8                                     Control   1         2          4      8

                                                 Exposure Time (hours)                                                       Exposure Time (hours)
Brain Microvascular Endothelial Cells (bEnd.3)




                             20                                                                          100
                                                                                                                                                     *




                                                                            (MCP-1 mRNA/GAPDH mRNA)
                                                                 *
(IL-6 mRNA/GAPDH mRNA)




                                                                                                         80




                                                                               Relative Fold Induction
   Relative Fold Induction




                             15                                                                                                               *

                                                                                                         60
                                                      *
                             10
                                                                        *                                40
                                                                                                                                   *

                              5
                                                                                                         20
                                            *
                                                                                                                         *
                              0                                                                           0
                                  Control   1         2          4      8                                      Control   1         2          4      8

                                                Exposure Time (hours)                                                        Exposure Time (hours)
Brain Microvascular Endothelial Cells (bEnd.3)




                               30                       *                                                   12
(E-selectin mRNA/GAPDH mRNA)




                                                                              (ICAM-1 mRNA/GAPDH mRNA)
                                                                                                                                                *
                               25




                                                                                  Relative Fold Induction
     Relative Fold Induction




                                                                   *                                         9
                               20                                                                                                                      *
                               15                                                                            6
                                                                          *
                               10
                                                                                                             3
                                5             *
                                0                                                                            0
                                    Control   1         2          4      8                                      Control   1         2          4      8

                                                  Exposure Time (hours)                                                        Exposure Time (hours)
Relative Fold Induction
              (TNF-Ī± mRNA/GAPDH mRNA)




          0
                 10
                       20
                             30
                                         40
                                                     50




Control
                                                     *




LPS
                  Relative Fold Induction
          0   (IL-1Ī² mRNA/GAPDH mRNA)
                 1
                      2
                             800
                                   900
                                              1000
                                                     1100
                                                            Microglia (BV-2)




Control
                                                *




LPS
Relative Fold Induction
               (IL-6 mRNA/GAPDH mRNA)




          0
                   1
                         2
                                   6500
                                               7000
                                                           7500
                                                                  8000




Control
                                                                  *




LPS
                 Relative Fold Induction
          0   (MCP-1 mRNA/GAPDH mRNA)
              10
                   20
                        30
                             40
                                  50
                                          60
                                                      70
                                                             80
                                                                  90
                                                                         Microglia (BV-2)




Control
                                                                  *




LPS
Relative Fold Induction
               (TNF-Ī± mRNA/GAPDH mRNA)




           0
                   1
                             2
                                           330
                                                  360
                                                        390
                                                              420




 Control
                                                              *




 LPS
                  Relative Fold Induction
               (IL-6 mRNA/GAPDH mRNA)
           0
               5
                   10
                        15
                                 20
                                      25
                                             30
                                                   35
                                                        40
                                                              45
                                                                    Astrocytes (C6)




Control
                                                              *




LPS
Relative Fold Induction
              (MCP-1 mRNA/GAPDH mRNA)




          0
               10
                    20
                         30
                              40
                                   50
                                        60
                                             70
                                                  80




Control
                                                  *
                                                       Astrocytes (C6)




LPS
In Vitro Ischemia/Reperfusion Model


A Novel Design of Double-
Layer Parallel-Plate Flow
Chamber & Its Biomedical
       Application
In Vitro Flow Chamber Systems




     Cone-Plate           Orbital Shaker




  Artificial Capillary   Parallel-Plate
Parallel-plate flow chambers (PPFC) have been
most commonly used for its simplicity of concept
                                                 x




             y
                                                 l
       h



                 w                        z




  Flow enters the parallel plates at the origin and exits
      where x equals the length of the chamber, l
Conventional PPFCs have shown weaknesses and
    problems in several aspects of its design
To eliminate these problems, we designed and
         developed a new double-layer PPFC
ā€¢ Accepts up to four glass slides facing each
  other so that the flow within the channel is
  exclusively formed by endothelial cells.
ā€¢ Provides a total of 96 cm2 cell
  monolayer per chamber.


ā€¢ Placing glass slides in
  series shortens the
  duration of procedure.
ā€¢ The multilayer design only
  requires 2D cutting, which is
  easier and faster to manufacture
  and modify.
The system becomes much simpler with the new chamber.
The new double-layer PPFC consists of separate
  layers of different materials and thicknesses
                          Acrylic sheets of 0.08 inch thickness

                          Acrylic sheets of 0.5 inch thickness



                         Each acrylic layer was cut
                         by Laser Computer Aided
                         Modeling and Manufacture
                         (LaserCAMM) machine.

                         The system is a
                         computerized laser cutter
                         that uses a laser beam to
                         cut sheet materials into
                         intricate patterns with a
                         high degree of accuracy.
The new double-layer PPFC consists of separate
  layers of different materials and thicknesses
                          Silicone gaskets of 0.03 inch thickness

                          Silicone gasket of 0.01 inch thickness




                         Silicone gaskets of 0.03
                         inch thickness serve as a
                         firm grip for glass slides.

                         The silicone gasket in the
                         middle constitutes the
                         channel height, h, and the
                         width, w.
The new double-layer PPFC consists of separate
  layers of different materials and thicknesses
                          Glass slides to fill up space

                          Glass slides which will have
                          cells seeded

                         Up to four glass slides can
                         be entered in a chamber.
                         Glass slides or
                         endothelial monolayers
                         are placed between the
                         gasket in the middle.
                         Placing endothelial
                         monolayers on both sides
                         of channel minimizes
                         pressure loss while having
                         a larger effective area.
The new double-layer PPFC consists of separate
  layers of different materials and thicknesses

                         Media enters through
                         the inlets.
                         Fills up a small reservoir
                         formed in the gasket.
                         Spreads evenly across
                         width through a thin slit.
                         Flows across the
                         endothelial monolayer.
                         Escapes the chamber
                         through the thin slit, the
                         small reservoir, and the
                         outlets.
The new double-layer PPFC consists of separate
  layers of different materials and thicknesses
                    To set up the chamber bubble-free,
                    the layers are installed in the order
                    from the bottom to the top layers
                    where the flow channel, reservoirs
                    are filled up with media by means of
                    syringe as each layer is piled up.
A flow loop system provides a constant
    hydrostatic pressure to the PPFC


                             Upper
                            Reservoir
                                             Flow

        Flow
        Meter

                                   Peristaltic
                                     Pump


                 Lower
 PPFC           Reservoir
ā€¢ The streamlines near the lateral walls were not disturbed
  ensuring that the lateral wall effects are negligible.
ā€¢ The chamber clearly applies a uniform magnitude of shear
  stress throughout the entire surface where endothelial cell
  monolayer will be placed.
RBE4   HMEC-1




Static




Flow
1.4
                            1.2                                                       Static
                                                                              1.2     Flow




                                                  (Relative Fold Induction)
(Relative Fold Induction)
  IL-6 Gene Expression




                                                      Gene Expression
                            0.9                                               1.0

                                                                              0.8
                            0.6
                                                                                         *                    *
                                                                              0.6
                                            *
                                                                              0.4
                            0.3
                                                                                                  *
                                                                              0.2

                            0.0                                               0.0
                                  Static   Flow                                     ICAM-1     VCAM-1   E-selectin
In Vitro Ischemia/Reperfusion Model
                              7
                                        Normal Flow

  (IL-6 mRNA/Ī²-Actin mRNA)
    Relative Fold Induction
                              6         Ischemia/Reperfusion                *
                              5

                              4

                              3

                              2

                              1

                              0
                                  0.5            1             6       12        24 (hours)

                                    Ischemia                       Reperfusion
In Vitro Ischemia/Reperfusion Model
                              5
                                        Normal Flow                         *
  (MCP-1 mRNA/Ī²-Actin mRNA)
                                        Ischemia/Reperfusion
                              4                                                    *
    Relative Fold Induction



                              3


                              2


                              1


                              0
                                  0.5            1             6       12        24 (hours)

                                    Ischemia                       Reperfusion
In Vitro Ischemia/Reperfusion Model
                               3


  (ICAM-1 mRNA/Ī²-Actin mRNA)
                                         Normal Flow
                                         Ischemia/Reperfusion
                                                                             *
     Relative Fold Induction


                               2




                               1




                               0
                                   0.5            1             6       12        24 (hours)

                                     Ischemia                       Reperfusion
In Vitro Ischemia/Reperfusion Model
                               6


  (VCAM-1 mRNA/Ī²-Actin mRNA)
                                         Normal Flow
     Relative Fold Induction
                               5
                                         Ischemia/Reperfusion             *

                               4

                               3
                                                                                    *
                               2

                               1

                               0
                                   0.5            1             6       12        24 (hours)

                                     Ischemia                       Reperfusion
In Vitro Ischemia/Reperfusion Model
                                   5


  (E-selectin mRNA/Ī²-Actin mRNA)
                                             Normal Flow
                                             Ischemia/Reperfusion
                                                                              *
                                   4                                                    *
       Relative Fold Induction



                                   3


                                   2


                                   1


                                   0
                                       0.5            1             6       12        24 (hours)

                                         Ischemia                       Reperfusion

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Nanotechnology Bio

  • 2. What is Nanotechnology? The creation of useful, functional materials, devices, and systems through: 1. Controlling and manipulating matter on the nanometer-length scale (1-100 nm), and 2. Exploiting novel phenomena and properties (physical, chemical, biological, mechanical, electrical) at the nanoscale. ā€œGoing Small for Big Advancesā€ ā€œGoing Small for Big Advancesā€
  • 3. Potential market for nanotechnology ? $1 trillion business within the next 10 to 15 years.
  • 4. Nanotechnology in Medicine ā€¢ Biological imaging for medical diagnostics. ā€¢ Advanced drug delivery systems. ā€¢ Biosensors for airborne chemicals or other toxins. ā€¢ Regenerative medicine: More durable, rejection-resistant artificial tissues and organs. NANOMEDICINE NANOMEDICINE
  • 5. Targeted Drug Delivery J Leukoc Biol 2005;78:585
  • 6. Cardiovascular Obesity / Disease Diabetes Risk Factors Inflammation Nanotechnology Tumor Neurological Angiogenesis Disorder / Metastasis
  • 7. Development of In Vivo and In Vitro Inflammatory Disease Models
  • 8. In Vivo Brain Inflammation Models ā€¢ Environmental Toxicants-Mediated Brain Inflammation. ā€¢ Bacterial Infection-Mediated Brain Inflammation.
  • 9. Environmental Toxicants & Brain Inflammation Isolation of brain regions C57/BL6 Mouse Determination of pro-inflammatory mediators - Cytokines: TNF-Ī±, IL-1Ī², IL-6 - Chemokine: MCP-1 Chlorpyrifos - Adhesion molecules: E-selectin, ICAM-1, VCAM-1 [OP Pesticide]
  • 10. 20 * Control (TNF-Ī± mRNA/GAPDH mRNA) CPF, 8 h CPF, 16 h Relative Fold Induction 15 CPF, 24 h * 10 * 5 * * * * * * * 0 HIP COR STR CER
  • 11. 25 (TNF-Ī± mRNA/GAPDH mRNA) Control CPF, 20 mg/kg * CPF, 70 mg/kg 20 Relative Fold Induction CPF, 140 mg/kg 15 10 * * * 5 * * * 0 HIP COR STR CER
  • 12. 6 Control * CPF, 4 h (IL-6 mRNA/GAPDH mRNA) 5 CPF, 8 h Relative Fold Induction CPF, 24 h 4 * * 3 * * * 2 * * 1 0 HIP COR STR CER
  • 13. 7 Control (IL-6 mRNA/GAPDH mRNA) CPF, 20 mg/kg * 6 CPF, 70 mg/kg Relative Fold Induction CPF, 140 mg/kg 5 4 * * 3 * * 2 1 0 HIP COR STR CER
  • 14. 6 * Control (MCP-1 mRNA/GAPDH mRNA) CPF, 8 h 5 CPF, 16 h Relative Fold Induction CPF, 24 h 4 * * 3 * * * 2 1 0 HIP STR CER
  • 15. 5 (MCP-1 mRNA/GAPDH mRNA) Control * CPF, 20 mg/kg * CPF, 70 mg/kg 4 Relative Fold Induction CPF, 140 mg/kg 3 * * 2 1 0 HIP STR CER
  • 16. 3.0 * (E-selectin mRNA/GAPDH mRNA) Control * CPF, 70 mg/kg 2.5 Relative Fold Induction * 2.0 1.5 1.0 0.5 0.0 HIP COR STR CER
  • 17. Bacterial Infection & Brain Inflammation Isolation of brain regions C57/BL6 Mouse Determination of pro-inflammatory mediators - Cytokines: TNF-Ī±, IL-1Ī², IL-6 - Chemokine: MCP-1 Lipopolysaccharide - Adhesion molecules: E-selectin, ICAM-1, VCAM-1 [LPS]
  • 18. 40 30 * Control (TNF-Ī± mRNA/GAPDH mRNA) 35 LPS * 25 Relative Fold Induction * 30 * * 20 25 * 20 15 15 10 10 5 5 0 0 HIP COR STR CER HIP COR STR CER 4 h Exposure 24 h Exposure
  • 19. 35 12 (IL-6 mRNA/GAPDH mRNA) 30 Control * * LPS Relative Fold Induction 25 9 20 * 6 15 * 10 * 3 * 5 0 0 HIP COR STR CER HIP COR STR CER 4 h Exposure 24 h Exposure
  • 20. 25 6 * Control * (IL-1Ī² mRNA/GAPDH mRNA) LPS 5 20 Relative Fold Induction 4 * 15 3 * * 10 * 2 * 5 1 0 0 HIP COR STR CER HIP COR STR CER 4 h Exposure 24 h Exposure
  • 21. 120 20 Control (MCP-1 mRNA/GAPDH mRNA) LPS * * 100 Relative Fold Induction * 15 80 * * 60 10 40 * * 5 20 0 0 HIP COR STR CER HIP COR STR CER 4 h Exposure 24 h Exposure
  • 22. 15 Control 10 (ICAM-1 mRNA/GAPDH mRNA) * LPS * * Relative Fold Induction 12 * 8 9 6 * * 6 * 4 * 3 2 0 0 HIP COR STR CER HIP COR STR CER 4 h Exposure 24 h Exposure
  • 23. 20 15 (E-selectin mRNA/GAPDH mRNA) Control LPS * * Relative Fold Induction 12 15 * * 9 * 10 * * 6 5 * 3 0 0 HIP COR STR CER HIP COR STR CER 4 h Exposure 24 h Exposure
  • 24. In Vitro Brain Inflammation Models ā€¢ Brain Microvascular Endothelial Cells (BMEC) ā€¢ Astrocytes ā€¢ Microglia
  • 25. Pro-inflammatory Stimuli-Mediated Inflammation in Brain Cells Pro-inflammatory Stimuli [100 ng/ml of LPS] Culture Media Brain Cells Determination of pro-inflammatory mediators - Cytokines: TNF-Ī±, IL-1Ī², IL-6 - Chemokine: MCP-1 - Adhesion molecules: E-selectin, ICAM-1
  • 26. Brain Microvascular Endothelial Cells (bEnd.3) 120 10 * (TNF-Ī± mRNA/GAPDH mRNA) (IL-1Ī² mRNA/GAPDH mRNA) 100 * 8 Relative Fold Induction Relative Fold Induction 80 6 60 * 4 40 * 2 20 * * 0 0 Control 1 2 4 8 Control 1 2 4 8 Exposure Time (hours) Exposure Time (hours)
  • 27. Brain Microvascular Endothelial Cells (bEnd.3) 20 100 * (MCP-1 mRNA/GAPDH mRNA) * (IL-6 mRNA/GAPDH mRNA) 80 Relative Fold Induction Relative Fold Induction 15 * 60 * 10 * 40 * 5 20 * * 0 0 Control 1 2 4 8 Control 1 2 4 8 Exposure Time (hours) Exposure Time (hours)
  • 28. Brain Microvascular Endothelial Cells (bEnd.3) 30 * 12 (E-selectin mRNA/GAPDH mRNA) (ICAM-1 mRNA/GAPDH mRNA) * 25 Relative Fold Induction Relative Fold Induction * 9 20 * 15 6 * 10 3 5 * 0 0 Control 1 2 4 8 Control 1 2 4 8 Exposure Time (hours) Exposure Time (hours)
  • 29. Relative Fold Induction (TNF-Ī± mRNA/GAPDH mRNA) 0 10 20 30 40 50 Control * LPS Relative Fold Induction 0 (IL-1Ī² mRNA/GAPDH mRNA) 1 2 800 900 1000 1100 Microglia (BV-2) Control * LPS
  • 30. Relative Fold Induction (IL-6 mRNA/GAPDH mRNA) 0 1 2 6500 7000 7500 8000 Control * LPS Relative Fold Induction 0 (MCP-1 mRNA/GAPDH mRNA) 10 20 30 40 50 60 70 80 90 Microglia (BV-2) Control * LPS
  • 31. Relative Fold Induction (TNF-Ī± mRNA/GAPDH mRNA) 0 1 2 330 360 390 420 Control * LPS Relative Fold Induction (IL-6 mRNA/GAPDH mRNA) 0 5 10 15 20 25 30 35 40 45 Astrocytes (C6) Control * LPS
  • 32. Relative Fold Induction (MCP-1 mRNA/GAPDH mRNA) 0 10 20 30 40 50 60 70 80 Control * Astrocytes (C6) LPS
  • 33. In Vitro Ischemia/Reperfusion Model A Novel Design of Double- Layer Parallel-Plate Flow Chamber & Its Biomedical Application
  • 34. In Vitro Flow Chamber Systems Cone-Plate Orbital Shaker Artificial Capillary Parallel-Plate
  • 35. Parallel-plate flow chambers (PPFC) have been most commonly used for its simplicity of concept x y l h w z Flow enters the parallel plates at the origin and exits where x equals the length of the chamber, l
  • 36. Conventional PPFCs have shown weaknesses and problems in several aspects of its design
  • 37. To eliminate these problems, we designed and developed a new double-layer PPFC ā€¢ Accepts up to four glass slides facing each other so that the flow within the channel is exclusively formed by endothelial cells. ā€¢ Provides a total of 96 cm2 cell monolayer per chamber. ā€¢ Placing glass slides in series shortens the duration of procedure. ā€¢ The multilayer design only requires 2D cutting, which is easier and faster to manufacture and modify.
  • 38. The system becomes much simpler with the new chamber.
  • 39. The new double-layer PPFC consists of separate layers of different materials and thicknesses Acrylic sheets of 0.08 inch thickness Acrylic sheets of 0.5 inch thickness Each acrylic layer was cut by Laser Computer Aided Modeling and Manufacture (LaserCAMM) machine. The system is a computerized laser cutter that uses a laser beam to cut sheet materials into intricate patterns with a high degree of accuracy.
  • 40. The new double-layer PPFC consists of separate layers of different materials and thicknesses Silicone gaskets of 0.03 inch thickness Silicone gasket of 0.01 inch thickness Silicone gaskets of 0.03 inch thickness serve as a firm grip for glass slides. The silicone gasket in the middle constitutes the channel height, h, and the width, w.
  • 41. The new double-layer PPFC consists of separate layers of different materials and thicknesses Glass slides to fill up space Glass slides which will have cells seeded Up to four glass slides can be entered in a chamber. Glass slides or endothelial monolayers are placed between the gasket in the middle. Placing endothelial monolayers on both sides of channel minimizes pressure loss while having a larger effective area.
  • 42. The new double-layer PPFC consists of separate layers of different materials and thicknesses Media enters through the inlets. Fills up a small reservoir formed in the gasket. Spreads evenly across width through a thin slit. Flows across the endothelial monolayer. Escapes the chamber through the thin slit, the small reservoir, and the outlets.
  • 43. The new double-layer PPFC consists of separate layers of different materials and thicknesses To set up the chamber bubble-free, the layers are installed in the order from the bottom to the top layers where the flow channel, reservoirs are filled up with media by means of syringe as each layer is piled up.
  • 44. A flow loop system provides a constant hydrostatic pressure to the PPFC Upper Reservoir Flow Flow Meter Peristaltic Pump Lower PPFC Reservoir
  • 45. ā€¢ The streamlines near the lateral walls were not disturbed ensuring that the lateral wall effects are negligible. ā€¢ The chamber clearly applies a uniform magnitude of shear stress throughout the entire surface where endothelial cell monolayer will be placed.
  • 46. RBE4 HMEC-1 Static Flow
  • 47. 1.4 1.2 Static 1.2 Flow (Relative Fold Induction) (Relative Fold Induction) IL-6 Gene Expression Gene Expression 0.9 1.0 0.8 0.6 * * 0.6 * 0.4 0.3 * 0.2 0.0 0.0 Static Flow ICAM-1 VCAM-1 E-selectin
  • 48. In Vitro Ischemia/Reperfusion Model 7 Normal Flow (IL-6 mRNA/Ī²-Actin mRNA) Relative Fold Induction 6 Ischemia/Reperfusion * 5 4 3 2 1 0 0.5 1 6 12 24 (hours) Ischemia Reperfusion
  • 49. In Vitro Ischemia/Reperfusion Model 5 Normal Flow * (MCP-1 mRNA/Ī²-Actin mRNA) Ischemia/Reperfusion 4 * Relative Fold Induction 3 2 1 0 0.5 1 6 12 24 (hours) Ischemia Reperfusion
  • 50. In Vitro Ischemia/Reperfusion Model 3 (ICAM-1 mRNA/Ī²-Actin mRNA) Normal Flow Ischemia/Reperfusion * Relative Fold Induction 2 1 0 0.5 1 6 12 24 (hours) Ischemia Reperfusion
  • 51. In Vitro Ischemia/Reperfusion Model 6 (VCAM-1 mRNA/Ī²-Actin mRNA) Normal Flow Relative Fold Induction 5 Ischemia/Reperfusion * 4 3 * 2 1 0 0.5 1 6 12 24 (hours) Ischemia Reperfusion
  • 52. In Vitro Ischemia/Reperfusion Model 5 (E-selectin mRNA/Ī²-Actin mRNA) Normal Flow Ischemia/Reperfusion * 4 * Relative Fold Induction 3 2 1 0 0.5 1 6 12 24 (hours) Ischemia Reperfusion