Bile duct on a chip: engineering a microfluidic platform for studying biliary epithelium in a dish

30 Jun 2022, 12:10
10m
Room: S3 A

Room: S3 A

Speaker

Willemse, Jorke (Department of Surgery, Erasmus MC Transplant Institute, University Medical Center, Rotterdam, )

Description

"Background and Aims:
Biliary complications that may arise after liver transplantation, such as non-anastomotic strictures and diffuse bile leakage, are challenging and complex. Ischemia-related cell death and impaired regeneration of damaged biliary epithelium is known to be involved in causing these complications. Intrahepatic cholangiocyte organoids (ICO) allow for the expansion and study of cholangiocyte-like cells, but access to the lumen of the organoids is limited and can only be studied after disrupting the 3D structure. There are currently no in vitro models mimicking the circumstances as exposure of bile ducts to warm ischemia time or cold storage, and therefore we aimed to establish a microfluidic bile-duct-on-chip (BDOC) platform for studying the effect of these conditions on biliary epithelium in vitro

Method:
ICO were initiated from human liver biopsies (N=5) obtained during liver transplant procedures. Three-channel BDOC (dimensions; length 1cm, width and height 500µm) were prepared by casting polydimethylsiloxane (PDMS) into a mold. Subsequently, plasma treated PDMS chips were bonded to glass slides. The BDOC channels were filled with solubilized decellularized human liver extracellular matrix and collagen type I pre-gel. Viscous finger patterning procedures were used to create channels inside these hydrogels. ICO-derived cells (50∙103 cells/channel) were introduced into the channels and ICO expansion medium was added to the reservoirs. The BDOC were incubated for up to 21 days. Growth of epithelial cells was monitored using confocal microscopy and histology.

Results:
ICO-derived cells populated the entire surface of the channels with a single layer of cells within 7 days after seeding. Whole mount confocal imaging revealed that cells were columnar in shape and morphologically looked like biliary epithelium. Zonula Occludens-1 (ZO-1) staining showed cholangiocyte-like polarization of cells in honey comb patterns. The cells express the cholangiocyte markers cytokeratin 7 and 19 on gene and protein level. Moreover, presence of glycocalyx components, such as fucosyl and sialic was confirmed based on histochemistry. This indicates that ICO form functional barriers.

Conclusion:
The results show that microfluidic approaches combined with cholangiocyte-like (KRT 7 and 19-positive) cells from ICO can be used to create healthy small diameter intrahepatic bile duct structures in vitro. This BDOC can thus serve as a platform to study epithelial damage and regeneration during warm and cold ischemic conditions in more detail in vitro. The platform can also be used to study the onset and progression of biliary diseases. <!--[if gte mso 9]><xml>
<w:LatentStyles DefLockedState=""false"" DefUnhideWhenUsed=""false"" DefSemiHidden=""false"" DefQFormat=""false"" DefPriority=""99"" LatentStyleCount=""371"">
<w:LsdException Locked=""false"" Priority=""0"" QFormat=""true"" Name=""Normal""/>
<w:LsdException Locked=""false"" Priority=""9"" QFormat=""true"" Name=""heading 1""/>
<w:LsdException Locked=""false"" Priority=""9"" SemiHidden=""true"" UnhideWhenUsed=""true"" QFormat=""true"" Name=""heading 2""/>
<w:LsdException Locked=""false"" Priority=""9"" SemiHidden=""true"" UnhideWhenUsed=""true"" QFormat=""true"" Name=""heading 3""/>
<w:LsdException Locked=""false"" Priority=""9"" SemiHidden=""true"" UnhideWhenUsed=""true"" QFormat=""true"" Name=""heading 4""/>
<w:LsdException Locked=""false"" Priority=""9"" SemiHidden=""true"" UnhideWhenUsed=""true"" QFormat=""true"" Name=""heading 5""/>
<w:LsdException Locked=""false"" Priority=""9"" SemiHidden=""true"" UnhideWhenUsed=""true"" QFormat=""true"" Name=""heading 6""/>
<w:LsdException Locked=""false"" Priority=""9"" SemiHidden=""true"" UnhideWhenUsed=""true"" QFormat=""true"" Name=""heading 7""/>
<w:LsdException Locked=""false"" Priority=""9"" SemiHidden=""true"" UnhideWhenUsed=""true"" QFormat=""true"" Name=""heading 8""/>
<w:LsdException Locked=""false"" Priority=""9"" SemiHidden=""true"" UnhideWhenUsed=""true"" QFormat=""true"" Name=""heading 9""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""index 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""index 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""index 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""index 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""index 5""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""index 6""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""index 7""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""index 8""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""index 9""/>
<w:LsdException Locked=""false"" Priority=""39"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""toc 1""/>
<w:LsdException Locked=""false"" Priority=""39"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""toc 2""/>
<w:LsdException Locked=""false"" Priority=""39"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""toc 3""/>
<w:LsdException Locked=""false"" Priority=""39"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""toc 4""/>
<w:LsdException Locked=""false"" Priority=""39"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""toc 5""/>
<w:LsdException Locked=""false"" Priority=""39"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""toc 6""/>
<w:LsdException Locked=""false"" Priority=""39"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""toc 7""/>
<w:LsdException Locked=""false"" Priority=""39"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""toc 8""/>
<w:LsdException Locked=""false"" Priority=""39"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""toc 9""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Normal Indent""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""footnote text""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""annotation text""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""header""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""footer""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""index heading""/>
<w:LsdException Locked=""false"" Priority=""35"" SemiHidden=""true"" UnhideWhenUsed=""true"" QFormat=""true"" Name=""caption""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""table of figures""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""envelope address""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""envelope return""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""footnote reference""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""annotation reference""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""line number""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""page number""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""endnote reference""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""endnote text""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""table of authorities""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""macro""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""toa heading""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Bullet""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Number""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List 5""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Bullet 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Bullet 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Bullet 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Bullet 5""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Number 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Number 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Number 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Number 5""/>
<w:LsdException Locked=""false"" Priority=""10"" QFormat=""true"" Name=""Title""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Closing""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Signature""/>
<w:LsdException Locked=""false"" Priority=""1"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Default Paragraph Font""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Body Text""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Body Text Indent""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Continue""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Continue 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Continue 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Continue 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""List Continue 5""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Message Header""/>
<w:LsdException Locked=""false"" Priority=""11"" QFormat=""true"" Name=""Subtitle""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Salutation""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Date""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Body Text First Indent""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Body Text First Indent 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Note Heading""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Body Text 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Body Text 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Body Text Indent 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Body Text Indent 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Block Text""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Hyperlink""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""FollowedHyperlink""/>
<w:LsdException Locked=""false"" Priority=""22"" QFormat=""true"" Name=""Strong""/>
<w:LsdException Locked=""false"" Priority=""20"" QFormat=""true"" Name=""Emphasis""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Document Map""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Plain Text""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""E-mail Signature""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""HTML Top of Form""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""HTML Bottom of Form""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Normal (Web)""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""HTML Acronym""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""HTML Address""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""HTML Cite""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""HTML Code""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""HTML Definition""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""HTML Keyboard""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""HTML Preformatted""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""HTML Sample""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""HTML Typewriter""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""HTML Variable""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Normal Table""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""annotation subject""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""No List""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Outline List 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Outline List 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Outline List 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Simple 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Simple 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Simple 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Classic 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Classic 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Classic 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Classic 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Colorful 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Colorful 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Colorful 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Columns 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Columns 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Columns 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Columns 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Columns 5""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Grid 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Grid 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Grid 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Grid 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Grid 5""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Grid 6""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Grid 7""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Grid 8""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table List 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table List 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table List 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table List 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table List 5""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table List 6""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table List 7""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table List 8""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table 3D effects 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table 3D effects 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table 3D effects 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Contemporary""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Elegant""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Professional""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Subtle 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Subtle 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Web 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Web 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Web 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Balloon Text""/>
<w:LsdException Locked=""false"" Priority=""39"" Name=""Table Grid""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Table Theme""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" Name=""Placeholder Text""/>
<w:LsdException Locked=""false"" Priority=""1"" QFormat=""true"" Name=""No Spacing""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading Accent 1""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List Accent 1""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid Accent 1""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1 Accent 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" Name=""Revision""/>
<w:LsdException Locked=""false"" Priority=""34"" QFormat=""true"" Name=""List Paragraph""/>
<w:LsdException Locked=""false"" Priority=""29"" QFormat=""true"" Name=""Quote""/>
<w:LsdException Locked=""false"" Priority=""30"" QFormat=""true"" Name=""Intense Quote""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List Accent 1""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading Accent 1""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List Accent 1""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid Accent 1""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading Accent 2""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List Accent 2""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid Accent 2""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List Accent 2""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading Accent 2""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List Accent 2""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid Accent 2""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading Accent 3""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List Accent 3""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid Accent 3""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List Accent 3""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading Accent 3""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List Accent 3""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid Accent 3""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading Accent 4""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List Accent 4""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid Accent 4""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List Accent 4""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading Accent 4""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List Accent 4""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid Accent 4""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading Accent 5""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List Accent 5""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid Accent 5""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List Accent 5""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading Accent 5""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List Accent 5""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid Accent 5""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading Accent 6""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List Accent 6""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid Accent 6""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List Accent 6""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading Accent 6""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List Accent 6""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid Accent 6""/>
<w:LsdException Locked=""false"" Priority=""19"" QFormat=""true"" Name=""Subtle Emphasis""/>
<w:LsdException Locked=""false"" Priority=""21"" QFormat=""true"" Name=""Intense Emphasis""/>
<w:LsdException Locked=""false"" Priority=""31"" QFormat=""true"" Name=""Subtle Reference""/>
<w:LsdException Locked=""false"" Priority=""32"" QFormat=""true"" Name=""Intense Reference""/>
<w:LsdException Locked=""false"" Priority=""33"" QFormat=""true"" Name=""Book Title""/>
<w:LsdException Locked=""false"" Priority=""37"" SemiHidden=""true"" UnhideWhenUsed=""true"" Name=""Bibliography""/>
<w:LsdException Locked=""false"" Priority=""39"" SemiHidden=""true"" UnhideWhenUsed=""true"" QFormat=""true"" Name=""TOC Heading""/>
<w:LsdException Locked=""false"" Priority=""41"" Name=""Plain Table 1""/>
<w:LsdException Locked=""false"" Priority=""42"" Name=""Plain Table 2""/>
<w:LsdException Locked=""false"" Priority=""43"" Name=""Plain Table 3""/>
<w:LsdException Locked=""false"" Priority=""44"" Name=""Plain Table 4""/>
<w:LsdException Locked=""false"" Priority=""45"" Name=""Plain Table 5""/>
<w:LsdException Locked=""false"" Priority=""40"" Name=""Grid Table Light""/>
<w:LsdException Locked=""false"" Priority=""46"" Name=""Grid Table 1 Light""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark""/>
<w:LsdException Locked=""false"" Priority=""51"" Name=""Grid Table 6 Colorful""/>
<w:LsdException Locked=""false"" Priority=""52"" Name=""Grid Table 7 Colorful""/>
<w:LsdException Locked=""false"" Priority=""46"" Name=""Grid Table 1 Light Accent 1""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark Accent 1""/>
<w:LsdException Locked=""false"" Priority=""51"" Name=""Grid Table 6 Colorful Accent 1""/>
<w:LsdException Locked=""false"" Priority=""52"" Name=""Grid Table 7 Colorful Accent 1""/>
<w:LsdException Locked=""false"" Priority=""46"" Name=""Grid Table 1 Light Accent 2""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark Accent 2""/>
<w:LsdException Locked=""false"" Priority=""51"" Name=""Grid Table 6 Colorful Accent 2""/>
<w:LsdException Locked=""false"" Priority=""52"" Name=""Grid Table 7 Colorful Accent 2""/>
<w:LsdException Locked=""false"" Priority=""46"" Name=""Grid Table 1 Light Accent 3""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark Accent 3""/>
<w:LsdException Locked=""false"" Priority=""51"" Name=""Grid Table 6 Colorful Accent 3""/>
<w:LsdException Locked=""false"" Priority=""52"" Name=""Grid Table 7 Colorful Accent 3""/>
<w:LsdException Locked=""false"" Priority=""46"" Name=""Grid Table 1 Light Accent 4""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark Accent 4""/>
<w:LsdException Locked=""false"" Priority=""51"" Name=""Grid Table 6 Colorful Accent 4""/>
<w:LsdException Locked=""false"" Priority=""52"" Name=""Grid Table 7 Colorful Accent 4""/>
<w:LsdException Locked=""false"" Priority=""46"" Name=""Grid Table 1 Light Accent 5""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark Accent 5""/>
<w:LsdException Locked=""false"" Priority=""51"" Name=""Grid Table 6 Colorful Accent 5""/>
<w:LsdException Locked=""false"" Priority=""52"" Name=""Grid Table 7 Colorful Accent 5""/>
<w:LsdException Locked=""false"" Priority=""46"" Name=""Grid Table 1 Light Accent 6""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark Accent 6""/>
<w:LsdException Locked=""false"" Priority=""51"" Name=""Grid Table 6 Colorful Accent 6""/>
<w:LsdException Locked=""false"" Priority=""52"" Name=""Grid Table 7 Colorful Accent 6""/>
<w:LsdException Locked=""false"" Priority=""46"" Name=""List Table 1 Light""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""List Table 2""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""List Table 3""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""List Table 4""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""List Table 5 Dark""/>
<w:LsdException Locked=""false"" Priority=""51"" Name=""List Table 6 Colorful""/>
<w:LsdException Locked=""false"" Priority=""52"" Name=""List Table 7 Colorful""/>
<w:LsdException Locked=""false"" Priority=""46"" Name=""List Table 1 Light Accent 1""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""List Table 2 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""List Table 3 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""List Table 4 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""List Table 5 Dark Accent 1""/>
<w:LsdException Locked=""false"" Priority=""51"" Name=""List Table 6 Colorful Accent 1""/>
<w:LsdException Locked=""false"" Priority=""52"" Name=""List Table 7 Colorful Accent 1""/>
<w:LsdException Locked=""false"" Priority"

41883605555

Presentation materials

There are no materials yet.