Sep 11, 2020
Dural Cell Isolation
- 1University of California, San Francisco
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
Protocol Citation: Andrea Argouarch 2020. Dural Cell Isolation . protocols.io https://dx.doi.org/10.17504/protocols.io.8ghhtt6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 19, 2019
Last Modified: September 11, 2020
Protocol Integer ID: 28905
Abstract
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.
Materials
STEP MATERIALS
Instant Sealing Sterilization Pouches, 3.5 x 5 in.Thermo FisherCatalog #0181250
25mm coverslips roundCatalog #GG-25
Dumont #5 Forceps Fine Science ToolsCatalog #11251-30
Fine Scissors - Tungsten Carbide Fine Science ToolsCatalog #14568-12
Instant Sealing Sterilization Pouches, 3.5 x 9 in.Thermo FisherCatalog #0181251
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Nalgene Cryogenic VialsVWR International (Avantor)Catalog #66008-706
DMSO Bio-Max, Cell Culture GradebioworldCatalog #40470005-2
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
Protocol materials
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
Instant Sealing Sterilization Pouches, 3.5 x 9 in.Thermo FisherCatalog #0181251
Nalgene Cryogenic VialsVWR International (Avantor)Catalog #66008-706
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Instant Sealing Sterilization Pouches, 3.5 x 5 in.Thermo FisherCatalog #0181250
25mm coverslips roundCatalog #GG-25
Dumont #5 Forceps Fine Science ToolsCatalog #11251-30
Fine Scissors - Tungsten Carbide Fine Science ToolsCatalog #14568-12
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
DMSO Bio-Max, Cell Culture GradebioworldCatalog #40470005-2
Instant Sealing Sterilization Pouches, 3.5 x 5 in.Thermo FisherCatalog #0181250
25mm coverslips roundCatalog #GG-25
Dumont #5 Forceps Fine Science ToolsCatalog #11251-30
Fine Scissors - Tungsten Carbide Fine Science ToolsCatalog #14568-12
Instant Sealing Sterilization Pouches, 3.5 x 9 in.Thermo FisherCatalog #0181251
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Nalgene Cryogenic VialsVWR International (Avantor)Catalog #66008-706
DMSO Bio-Max, Cell Culture GradebioworldCatalog #40470005-2
Preparation for Isolation
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)
25mm coverslips roundCatalog #GG-25
b. Surgical Tools – 1 each per 23 cm pouch
i. Dumont # 5 Forceps
Dumont #5 Forceps Fine Science ToolsCatalog #11251-30
ii. Tungsten Scissors
Fine Scissors - Tungsten Carbide Fine Science ToolsCatalog #14568-12
c. Seal autoclave pouch and autoclave. Confirm that autoclave tape has turned black
Instant Sealing Sterilization Pouches, 3.5 x 9 in.Thermo FisherCatalog #0181251
Instant Sealing Sterilization Pouches, 3.5 x 5 in.Thermo FisherCatalog #0181250
Turn off UV lights and clean hood with 70% ethanol
Clean items with 70% ethanol and bring into hood
a. DPBS -/-
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
b. Sterile Filtered Media
i. High Glucose DMEM with Sodium Pyruvate (1X)
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
ii. Heat Inactivated FBS (10%)
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
iii. PenStrep (1%)
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
c. Biopsy in 50ml conical with ~20 mls 20 mL 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 3 mL of DPBS -/- each
f. 10 cm dish for rinsed biopsy, labeled ‘clean’, add ~15 mls 15 mL 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
Nalgene Cryogenic VialsVWR International (Avantor)Catalog #66008-706
i. Label: Date, ID, Dura Tissue
ii. Add 1 ml 1 mL of freezing media per vial
l. Freezing media
i.100% DMSO to a final of 10%
DMSO Bio-Max, Cell Culture GradebioworldCatalog #40470005-2
ii. 90% of media
Isolation
Isolation
Spray conical with biopsy into the hood and pour media with biopsy into the dirty 10 cm dish
With forceps, submerge biopsy in the 1st DPBS well, tilt, rinse and swirl for 1 min 00:01:00
With forceps, submerge biopsy in the 2nd DPBS well, tilt, rinse and swirl for 1 min 00:01:00
With forceps, submerge biopsy in the 3rd DPBS well, tilt, rinse and swirl for 1 min 00:01:00
With forceps, submerge biopsy in the 4th DPBS well, tilt, rinse and swirl for 1 min 00:01:00
With forceps, transfer the rinsed biopsy into the clean 10 cm dish with new media
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 -80 °C for 25-48hrs 48:00:00 , then transfer to ln2 -190 °C for long-term storage
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
Add 200 µl 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
Place a coverslip over each droplet, starting from one side, centering it, and dropping it into the well
a. Avoid air bubbles
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
Add 3 mls 3 mL 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
Observe tissue integrity under microscope
Gently place 6 well plate in incubator (5% CO2, 37oC) 37 °C
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 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
Aspirate biohazard media and throw away biohazard materials properly
Clean and sterilize hood with 70% ethanol and turn UV on. Update cell culture notes in lab notebook.