Jul 01, 2020
Cryopreservation of tissues for primary cell culture and single cell sequencing
- Harikrishna Nakshatri1
- 1Indiana University/Purdue University at Indianapolis
- Human Cell Atlas Method Development Community
Protocol Citation: Harikrishna Nakshatri 2020. Cryopreservation of tissues for primary cell culture and single cell sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bhvjj64n
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: June 24, 2020
Last Modified: July 01, 2020
Protocol Integer ID: 38539
Keywords: Cryopreservation, Primary Cell Culture, Single Cell Sequencing,
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Abstract
This protocol details the cryopreservation of tissues used for primary cell cultures and single cell sequencing.
Attachments
Guidelines
After you're done:
Thawing for single cell analyses or preparing for cells:
Unlike cooling, thawing has to be rapid at 37 °C and transfer the content to 10 mL warm media to wash tissues. Since DMSO is known to cause differentiation of stem cells, we wash tissues thoroughly before starting digestion (particularly for generating primary cell line).
Materials
PRIMARY CELL F12-DMEM (low glucose) 3:1 Media
1) F12 (Cat# 11765-054, Gibco) …………………………………………………………………….............. 375 ml
2) DMEM (low glucose, Cat# 12320-032, Gibco) ………………………………………………........ 125 ml
3) FBS (Cat# 26140-079, Gibco) ...……………………………………………………………………......... 25 ml
4) Hydrocortisone (Cat# H0888, Sigma-Aldrich, 0.4 µg/ml). The stock is 1mg/ml 200 µL
5) Penicillin-Streptomycin Solution, 100X (Cat# 30-002-CI, Corning) ......……….……… 5 ml
6) Insulin (Cat# I5500, Sigma-Aldrich, 5 µg/ml). The stock is 1 mg/ml ……………….... 2.5 ml
7) EGF (Cat# 236-EG-200, R&D systems, 20 ng/ml). The stock is 2 µg/µl………………… 5 µL
Note: To the all cells, also add the following during culturing and changing the media. For 10 ml media , use 40 µL of 6 mg/ml Adenine and 5 µL of 10 mM ROCK inhibitor Y-27632 .
1) Adenine (Cat# A-9001, Sigma-Aldrich). The stock is 6mg/ml …………………………………….................... 40 µL
2) ROCK inhibitor (Y-27632, Cat# ALX-270-333-M005, Enzo Life Sciences). The stock is 10 mM ..… 5 µL
Other Materials:
- Cryoprotective Freezing Medium (Lonza cat. no. 12-132A)
- CoolCell containers (Nalgene Cat#5100-0001)
Safety warnings
Please see the Safety Data Sheet (SDS) for any protocol hazards and warnings.
Freezing Protocol
Freezing Protocol
Collect tissues in the Cryoprotective Freezing Medium with ROCK inhibitor.
Mince tissues into small pieces.
Resuspend in 500 µL primary cell medium and 500 µL cryoprotective freezing medium + 0.5 µL ROCK inhibitor .
Aliquot into cryogenic storage vials.
Place vials in CoolCell containers.
Cells should be frozen slowly at 1 °C per minute .
Note
This can be achieved using the CoolCell containers at a -70 °C to -90 °C freezer overnight, then transferring to liquid nitrogen storage.