TITLE: Dural Cell Isolation 
AUTHORS: Andrea Argouarch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Isolation of cells from human dura mater. Protocol includes tissue freezing, cutting the tissue with surgical tools, plating into a 6 well plate, placing a coverslip on top of the tissue, and adding cell culture media.</div></div>

[STEPS]
?. [Preparation for Isolation]
Prepare solid autoclaving     a. 25 mm coverslips – 7 coverslips per 13 cm pouch (slide coverslips in the middle of the pouch before opening)    b. Surgical Tools – 1 each per 23 cm pouch        i. Dumont # 5 Forceps        ii.  Tungsten Scissors    c. Seal autoclave pouch and autoclave. Confirm that autoclave tape has turned black
?. [Preparation for Isolation]
Turn off UV lights and clean hood with 70% ethanol
?. [Preparation for Isolation]
Clean items with 70% ethanol and bring into hood    a. DPBS -/-     b. Sterile Filtered Media        i. High Glucose DMEM with Sodium Pyruvate (1X)         ii. Heat Inactivated FBS (10%)         iii. PenStrep (1%)     c. Biopsy in 50ml conical with ~20 mls   of media    d. 10 cm dish for biopsy and its media, labeled ‘dirty’    e. 6 well plate for washes, four wells labeled 1-4, add 3 mls   of DPBS -/- each    f. 10 cm dish for rinsed biopsy, labeled ‘clean’, add ~15 mls    of new fibroblast media. Make sure biopsy is submerged in media and will not dry out     g. 10 cm plate to hold tools labeled ‘tools’    h. 6 well plate, labeled with ID, date, and p0    i. Autoclaved coverslips    j. Autoclaved surgical tools    k.3-4 Ln2 vials  pre labeled for extra dural tissue        i. Label: Date, ID, Dura Tissue        ii. Add 1 ml  of freezing media per vial    l. Freezing media         i.100% DMSO to a final of 10%         ii. 90% of media
20 mL
3 mL
15 mL
1 mL
?. [Isolation]
Spray conical with biopsy into the hood and pour media with biopsy into the dirty 10 cm dish
?. [Isolation]
With forceps, submerge biopsy in the 1st DPBS well, tilt, rinse and swirl for 1 min
?. [Isolation]
With forceps, submerge biopsy in the 2nd DPBS well, tilt, rinse and swirl for 1 min
?. [Isolation]
With forceps, submerge biopsy in the 3rd DPBS well, tilt, rinse and swirl for 1 min
?. [Isolation]
With forceps, submerge biopsy in the 4th DPBS well, tilt, rinse and swirl for 1 min
?. [Isolation]
With forceps, transfer the rinsed biopsy into the clean 10 cm dish with new media
?. [Isolation]
Cut biopsy into 3-5 regions, freeze 2-4 sections and keep 1 section to culture     a. Place pieces into ln2 vials with tweezers, careful not to touch the outside of the vial    c. Place in a Mr. Frosty at -80C   for 25-48hrs  , then transfer to ln2   for long-term storage
-80 °C
-190 °C
?. [Isolation]
Cut the small section of biopsy into smaller pinhead sized pieces with forceps and scissors     a. Don’t let biopsy to dry out    b. Aim for 18-20 pieces depending on biopsy size     c. Once finished, cover dish and carefully move aside
?. [Isolation]
Add 200 µl   droplet of media in the center of each well of a 6 well plate    a. Place similar sized pieces of tissue within the middle of the droplet
200 µl
?. [Isolation]
Place a coverslip over each droplet, starting from one side, centering it, and dropping it into the well    a. Avoid air bubbles
?. [Isolation]
Gently press the coverslip down to make pieces adhere evenly and firmly to the bottom of the well     a. Muse sure the coverslips are firm, secure, flat, even, and not wobbling     b. Keep the pieces in the center    c. Tissue should be bit squished out flat, but still dense and compact
?. [Isolation]
Add 3 mls   of media per well, making sure that the coverslips does not rise by gently placing the forceps on top of coverslip. Add the media quickly to break the surface tension
3 mL
?. [Isolation]
Observe tissue integrity under microscope
?. [Isolation]
Gently place 6 well plate in incubator (5% CO2, 37oC)
37 °C
?. [Isolation]
Let the biopsy tissue settle for a week before feeding, and observe cell growth. Do not move plate for the first week.
?. [Clean Up]
Clean surgical tools, wear waterproof lab coat and eye protection/PPE    a. Brush and clean with 409 soap water. Rinse with water, dry on kimwipe, rinse with 100% ethanol, and then dry completely with kimwipe to prevent rust or water marks.     b. Prep for next autoclaving cycle
?. [Clean Up]
Aspirate biohazard media and throw away biohazard materials properly
?. [Clean Up]
Clean and sterilize hood with 70% ethanol and turn UV on. Update cell culture notes in lab notebook.