Cryopreservation of tissues for primary cell culture and single cell sequencing
Jul 01, 2020
Open access
Protocol CitationHarikrishna 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: Jun 24, 2020
Last Modified: Jul 01, 2020
PROTOCOL integer ID: 38539
Keywords: Cryopreservation, Primary Cell Culture, Single Cell Sequencing

Public workspaceCryopreservation of tissues for primary cell culture and single cell sequencing

  • Harikrishna Nakshatri1
  • 1Indiana University/Purdue University at Indianapolis
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Abstract
This protocol details the cryopreservation of tissues used for primary cell cultures and single cell sequencing.
Guidelines
After you're done:

Thawing for single cell analyses or preparing for cells:
Unlike cooling, thawing has to be rapid at Temperature37 °C and transfer the content to Amount10 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) …………………………………………………………………….............. Amount375 ml
2) DMEM (low glucose, Cat# 12320-032, Gibco) ………………………………………………........ Amount125 ml
3) FBS (Cat# 26140-079, Gibco) ...……………………………………………………………………......... Amount25 ml
4) Hydrocortisone (Cat# H0888, Sigma-Aldrich, 0.4 µg/ml). The stock is 1mg/ml Amount200 µL
5) Penicillin-Streptomycin Solution, 100X (Cat# 30-002-CI, Corning) ......……….……… Amount5 ml
6) Insulin (Cat# I5500, Sigma-Aldrich, 5 µg/ml). The stock is 1 mg/ml ……………….... Amount2.5 ml
7) EGF (Cat# 236-EG-200, R&D systems, 20 ng/ml). The stock is 2 µg/µl………………… Amount5 µL

Note: To the all cells, also add the following during culturing and changing the media. For Amount10 ml media , use Amount40 µL of 6 mg/ml Adenine and Amount5 µL of 10 mM ROCK inhibitor Y-27632 .
1) Adenine (Cat# A-9001, Sigma-Aldrich). The stock is 6mg/ml …………………………………….................... Amount40 µL
2) ROCK inhibitor (Y-27632, Cat# ALX-270-333-M005, Enzo Life Sciences). The stock is 10 mM ..… Amount5 µL

Other Materials:
  • Cryoprotective Freezing Medium (Lonza cat. no. 12-132A)
  • CoolCell containers (Nalgene Cat#5100-0001)
Safety warnings
Attention
Please see the Safety Data Sheet (SDS) for any protocol hazards and warnings.
Freezing Protocol
1
Collect tissues in the Cryoprotective Freezing Medium with ROCK inhibitor.
2
Mince tissues into small pieces.
3
Resuspend in Amount500 µL primary cell medium and Amount500 µL cryoprotective freezing medium + Amount0.5 µL ROCK inhibitor .
4
Pipetting
Aliquot into cryogenic storage vials.
5
Place vials in CoolCell containers.
6
Overnight
Cells should be frozen slowly at Temperature1 °C per minute .
Note
This can be achieved using the CoolCell containers at a Temperature-70 °C to Temperature-90 °C freezer overnight, then transferring to liquid nitrogen storage.