Sep 10, 2020
Freezing/Cell Banking
- 1University of California, San Francisco
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
Protocol Citation: Andrea Argouarch 2020. Freezing/Cell Banking. protocols.io https://dx.doi.org/10.17504/protocols.io.8gmhtu6
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 10, 2020
Protocol Integer ID: 28909
Abstract
Protocol includes banking dural cells into cryovials for long term storage in liquid nitrogen.
Materials
STEP MATERIALS
Mr. Frosty Freezing Container, 2mL tubes, Nalgene Mr. Frosty Freezing Container for 1-2mL cryogenic tubes, PC, clear w/ blue lid, 1/Cs.Thermo FisherCatalog #5100-0001
Cryo-TagsUSA ScientificCatalog #9187-1100
Trypan Blue Solution, 0.4%Thermo FisherCatalog #15250061
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
DMSO Bio-Max, Cell Culture GradebioworldCatalog #40470005-2
C-Chip™ Disposable Hemacytometers, Improved Neubauer; 50/Pk.Thermo FisherCatalog #22600100
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
Protocol materials
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Mr. Frosty Freezing Container, 2mL tubes, Nalgene Mr. Frosty Freezing Container for 1-2mL cryogenic tubes, PC, clear w/ blue lid, 1/Cs.Thermo FisherCatalog #5100-0001
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
Cryo-TagsUSA ScientificCatalog #9187-1100
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Trypan Blue Solution, 0.4%Thermo FisherCatalog #15250061
DMSO Bio-Max, Cell Culture GradebioworldCatalog #40470005-2
C-Chip™ Disposable Hemacytometers, Improved Neubauer; 50/Pk.Thermo FisherCatalog #22600100
Cryo-TagsUSA ScientificCatalog #9187-1100
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Trypan Blue Solution, 0.4%Thermo FisherCatalog #15250061
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
DMSO Bio-Max, Cell Culture GradebioworldCatalog #40470005-2
C-Chip™ Disposable Hemacytometers, Improved Neubauer; 50/Pk.Thermo FisherCatalog #22600100
Mr. Frosty Freezing Container, 2mL tubes, Nalgene Mr. Frosty Freezing Container for 1-2mL cryogenic tubes, PC, clear w/ blue lid, 1/Cs.Thermo FisherCatalog #5100-0001
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
Observations
Observations
At 100% confluency, take pictures of cell morphology, freeze down for banking (~0.5 Million per vial), collect remaining cells in flask for gDNA, and seed cells into 1xT25 for karyotyping (~30,000 cells, 60 ul)
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
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
c. Label 2xT150 flasks with ID, date, and p2, add 28 mls 28 mL of media per flask
d. 0.05% Trypsin
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
Preparation
Preparation
Turn off UV lights and clean hood with 70% ethanol
Labeling cryovials
a. Create labels using Microsoft Word, following the formatting instructions
Cryo-TagsUSA ScientificCatalog #9187-1100
b. Print out estimated number of labels for the both flasks (~15) with ID, date, p2, and ~0.5 mill cells.
c. Arrange cryovials in 96, 1.5ml tube holder with spaces in between each vial
d. Spray label sheets thoroughly with 70% ethanol, dry, and bring into hood to adhere onto each vial
e. Label top of each vial with ID
f. Unscrew cap in order to easily lift up when pipetting in cell suspension
Clean items with 70% ethanol and bring into hood
a. DPBS -/-
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
b. Sterile Filtered Media
DMEM, high glucose, pyruvateThermo FisherCatalog #11995073
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Fetal Bovine Serum VWR International (Avantor)Catalog #97068-091
c. 0.05% Trypsin
i. Aliquot to and warm before use
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
d. Freezing media (made fresh)
i. 100% DMSO to 10%
DMSO Bio-Max, Cell Culture GradebioworldCatalog #40470005-2
ii. 90% Media
e. Incyto disposable hemocytometers
C-Chip™ Disposable Hemacytometers, Improved Neubauer; 50/Pk.Thermo FisherCatalog #22600100
f. Trypan blue, add 40 ul 40 µL into a 1.5 ml tube
Trypan Blue Solution, 0.4%Thermo FisherCatalog #15250061
g. T25 labeled to send to Cell Line Genetics for karyotyping (KT)
h. 15 ml conical with 10 mls 10 mL of media to dilute KT cells
i. 15 ml conical to collect cells for gDNA
Culturing
Culturing
Take 3-4 BF images of morphology. Save TIFF under ID and passage number
Aspirate old media
Rinse by adding 5 mls 5 mL of DPBS per flask and gently swirling
Aspirate DPBS
Add 3 ml 3 mL of trypsin per flask and place in incubator 37 °C for 3 mins 00:03:00 until cells are detached
a. Can also gently tap the side of the flask to detach cells, check under microscope to confirm
Add 9 mls 9 mL of fibroblast media per flask to inactivate trypsin
Collect cell suspension in a 50 ml conical
Rinse flask with 12 mls 12 mL of PBS to collect remaining cells, place in 15ml conical for gDNA
Measure final cell suspension volume with serological and mix
Dilute 10 ul 10 µL of cell suspension into trypan blue, tap to mix
Load 10 ul 10 µL of trypan blue and cell dilution into the hemocytometer
Count non blue cells in 4 grind corners during spin and then calculate total cells and resuspension volume for 0.5E6 cells per ml
Total cells for 2 flasks = (Cell Count/4 grinds) x 5 dilution factor x 10,000 x ~25mls suspension volume
a. Resuspension volume for 0.5E6 cells per ml = Number of vials
Spin both conicals at 1000 rpm 1000 rpm for 5 mins 00:05:00 during counting
Aspirate supernatant for both pellets (freezing and gDNA), being careful to not aspirate the pellet
Tap the pellet to resuspend for freezing
Store gDNA pellet at -80C -80 °C until mycoplasma assay
Add freezing media dropwise to cell pellet and add 1 ml 1 mL per cryovial. Spin KT cell pellet during this time.
Prepare for KT in T25 flask
a. Take 60-100 ul 60 µL of DMSO cell suspension and dilute into 10 mls 10 mL of media
b. Spin for 1000 rpm 1000 rpm for 5 mins 00:05:00
c. Aspirate, resuspend in 5 ml 5 mL of media, and add to 1xT25 for KT
d. Place flask in incubator (5% CO2, 37oC) 37 °C
e. Send KT to Cell Line Genetics at 40-50% confluency in about 5-6 days
Secure each cryovial cap tightly and place in Mr. Frosty
Mr. Frosty Freezing Container, 2mL tubes, Nalgene Mr. Frosty Freezing Container for 1-2mL cryogenic tubes, PC, clear w/ blue lid, 1/Cs.Thermo FisherCatalog #5100-0001
Place Mr. Frosty in -80oC -80 °C for 24-48 hours 48:00:00 and then transfer to liquid nitrogen -190 °C
Freeze back up cells from 6 well plate, place in Mr. Frosty for 24-48 hours 48:00:00 , then transfer to LN2 -190 °C
Clean Up
Clean Up
Throw away biohazard materials properly
Clean and sterilize hood with 70% ethanol and turn on UV
Update cell culture notes in lab notebook