Endothelial Cell-Based Biosensor for

Alzheimer’s Disease Diagnosis

Trey Hope, Joyce Knezevic, Colton Kostelnik

BMEN 428
Problem Definition
● Alzheimer’s Disease (AD) affects over 5 million people over the age of
65 in the United States1.

● The aggregation of amyloid-β (Aβ), an oligomer, forms plaques in the

brain that are directly related to AD. The disease state of the brain
progresses as the Aβ plaques get larger.

● As the Aβ plaques get larger the integrity of the tight junctions in the
cells of the Blood Brain Barrier (BBB) decreases. This reduction in
integrity allows other toxic particles to enter the brain and can be
exploited to monitor the progression of AD2.

● Currently there is no available in-vivo method to monitor the

progression of AD.
Goals and Objectives
Goal 1: Learn cell culturing methods and protocols by culturing human umbilical cord vein cells (HUVEC)
● Objective 1: Culture cells to and split at ~80% confluency and optimize HUVEC cell culture protocol
● Objective 2: Seed cells on coverslips to determine proper seeding concentration
● Objective 3: Seed cells on cellulose acetate membrane and perform immunocytochemistry to determine if
cells are attached to the membrane

Goal 2: Culture HBMVEC on cellulose acetate membrane

● Objective 1: Seed cells on cellulose acetate membrane and culture to ~90% confluency
● Objective 2: Determine cells adhesion to cellulose acetate membrane through immunocytochemistry
● Objective 3: Perform immunocytochemistry on cells to determine presence of tight junction proteins through
presence of ZO-1 protein

Goal 3: Attach membrane with HBMVEC on sensor from Vector Inc

● Objective 1: Attach membrane to sensor
● Objective 2: Expose cells to Aβ and measure TEER values with sensor
Methodology
● Goal 1: HUVECs
○ Culture HUVECs for one week to reach ~80% confluency
○ Once we culture 0.5 x 10⁶ cell/mL, seed cells onto membrane, coverslip or vials
■ Culture membrane and coverslips in same 6 well plate to determine when membrane has reached
100% confluency
○ Utilize 2% gelatin solution to increase cell adherence on membrane
○ Immunocytochemistry
■ DAPI - Nucleus

● Goal 2: HBMVECs
○ Culture HBMVECs and seed onto the membranes and coverslips until ~90% confluent
■ Utilize hydrocortisone media to upregulate ZO-1 expression and induce tight junction formation
○ Immunocytochemistry
■ DAPI - Nucleus
■ Phalloidin - F-actin
■ Anti-ZO1 Antibody - Tight Junctions

● Goal 3: Implementation
○ Implement membrane with monolayer of HBMVECs onto Vector Inc. sensor
Culture Plan
Results: Immunocytochemistry 1

● Purpose: Follow staining protocol and obtain images of HUVEC

● Stained P12 HUVEC with ZO-1 antibodies, Phalloidin and DAPI
● Images showed ZO-1 on inside of cells rather than outside
○ Combination of moving TritonX before stainnig for ZO-1 and level of ZO-1 in HUVEC
● Small amount of cells on membrane
○ Cells may have been washed off during protocol
○ Membrane might need to be treated with attachment factors, gelatin or collagen
Results: Immunocytochemistry 1

These are the different channels from the

same coverslip that was treated with
regular media and seeded at 0.5 x 106
mg/mL

[A]: DAPI

[B]: Phalloidin

[C]: ZO-1

[D]: DAPI, Phalloidin and ZO-1

Results: Immunocytochemistry 1

These are two images from the

membrane that was treated with
regular media.

[A] & [C]: DAPI

[B] & [D]: ZO-1

Results: Immunocytochemistry 2
● Purpose
○ Upregulate ZO-1
○ Image ZO-1 in space between HUVEC cells
○ Image HUVEC on membrane
● Wells treated with hydrocortisone and media
○ Treated for 5 days
○ Decrease in cell count after day 4 due to overcrowding
● Stained Membranes with DAPI
● Stained coverslips with ZO-1 antibodies, Phalloidin and DAPI
● TritonX moved after ZO-1
● Received inconclusive results
Results: Immunocytochemistry 3
● Purpose: Reduce number of washing steps, upregulate ZO-1 in tight junctions
of HUVEC
● Two six well plates
○ 0.50 x 106 cells/mL and 0.40 x 106 cells/mL
■ Both controls had controls treated with regular media
● Stained one coverslip at 0.4 x 106 cells/mL and two 0.5 x 106 cells/mL
coverslips
○ Coverslips stained with ZO-1 antibodies and DAPI
 Results Immunocytochemistry 3

These are the images from the

hydrocortisone coverslips that were
treated with hydrocortisone media.

[A]: DAPI at 0.40 x 10⁶ cells/ mL

[B]: ZO-1 at 0.40 x 10⁶ cells/ mL

[C]: DAPI at 0.50 x 10⁶ cells/ mL

[D]: ZO-1 at 0.50 x 10⁶ cells/ mL

Results: Immunocytochemistry 3

These images show a comparison of ZO-1

set at the same LUT. Increase in brightness
between seeding concentrations and
treatment

[A]: Control at 0.40 x 10⁶ cells/ mL

[B]: Hydrocortisone at 0.40 x 10⁶ cells/ mL

[C]: Control at 0.50 x 10⁶ cells/ mL

[D]: Hydrocortisone at 0.50 x 10⁶ cells/ mL

Current Status
● P10 HUVEC
○ Stained control, one membrane with hydrocortisone media and one without hydrocortisone media
■ Purpose: Determine if HUVEC adhere to membrane and produce ZO-1
■ Have not collected images yet
● P11 HUVEC
○ Use weights to stick membranes to bottom of well plate and use gelatin to adhere HUVEC to membrane
Timeline
 Materials Extended Price Use

Budget Endothelial Cell Growth Medium

(4 x 500 ml)
$448 Culture medium for HUVEC cell line

Masconas ( 1 x 500 ml) $7.97 Washing excessive media off of cell during passaging and seeding

Trypsin (2 x 40 ml) $2.78 Detaches cells from surfaces during passaging and seeding

HBMVEC Growth Medium (500 mL) $170 Essential for the growth of cell cultures for proper experimentation.

HBMVEC Passage Reagents $136 Three different reagents used during splitting and seeding protocols

ZO-1 Monoclonal Antibody (ZO1-1A12) $188 Primary antibody that attaches to tight junction protein, ZO-1

Donkey anti-Mouse Alexa Fluor 488 $142 Secondary antibody that attaches to the primary and the
fluorochrome

Cy3, Hydrocortisone, DAPI, Attachment Provided by the previous year’s senior design group or Dr. Moss’s
Factors, Gelatin, RGD Peptides, CA Lab
membrane, HBMVECs

Total Price $1094.75 Additional funds provided by Dr. Moss Lab

(mius S,H,T)
Future Direction
Future Direction
● Test the use of attachment factors, collagen and gelatin with membrane for
better cell adhesion
○ Continue using HUVEC for testing purposes
○ Staining only with DAPI and Cytopainter Phalloidin

● Once protocols for membrane adhesion are viable, switch to HBMVEC

○ Optimize the protocols for HBMVEC and treat with Hydrocortisone media to upregulate ZO-1
○ Strain for DAPI, Cytopainter Phalloidin and ZO-1

● Continue HUVEC-membrane culturing for Vector’s sensor

○ Membrane may change due to new sensor
References
1. Jiménez N, Krouwer VJD, Post JA. 2013. A new, rapid and reproducible method to obtain high quality endothelium in vitro.
Cytotechnology. 65 (1):1-14.
2. Gonzalez-Velasquez FJ, Kotarek JA, Moss MA. 2008. Soluble aggregates of the amyloid-β protein selectively stimulate
permeability in human brain microvascular endothelial monolayers. Journal of Neurochemistry 107:466–477.
3. Morita K, Tsukita S, Miyachi Y. 2004. Tight junction-associated proteins (occludin, ZO-1, claudin-1, claudin-4) in squamous
cell carcinoma and Bowen’s disease. British Journal of Dermatology. 151 (2): 328-334.
4. Ettinger A, Wittmann T. 2014. Fluorescence Live Cell Imaging. Methods Cell Biology 123:77-94.
5. Gimbrone MA. 1974. HUMAN VASCULAR ENDOTHELIAL CELLS IN CULTURE: Growth and DNA Synthesis. The
Journal of Cell Biology 60:673–684.