Aug 15, 2020

Public workspace703.3 URMC HTC_Cryopreservation_of_Isolated_Cells_061220

DOI
dx.doi.org/10.17504/protocols.io.biz6kf9e
  • 1University of Rochester Medical Center
  • Human Cell Atlas Method Development Community
  • LungMap2 Consortium
Gloria S Pryhuber
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DOI: dx.doi.org/10.17504/protocols.io.biz6kf9e
Protocol Citation: Ravi Misra, Heidie Huyck, Gautam Bandyopadhyay, Gloria S Pryhuber 2020. 703.3 URMC HTC_Cryopreservation_of_Isolated_Cells_061220. protocols.io https://dx.doi.org/10.17504/protocols.io.biz6kf9e
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: July 25, 2020
Last Modified: August 15, 2020
Protocol Integer ID: 39710
Keywords: Cell cryopreservation, Thaw cryoprotected cells,
Abstract
Lung MAP HTC - BioRepository for Investigation of Neonatal Diseases of the Lung (BRINDL)

Standard Operating Procedures 703.2_HTC102314rev061019

Cryopreservation of Isolated Cells
Purpose and Scope of the Procedure or Laboratory Assay
  1. Stable frozen storage of isolated cells from lung, lymphoid tissues and blood while inducing least artifact possible
Guidelines
Scientific Principles
Cells may be frozen at a slow controlled rate of minus one degree Celsius per minute in cryoprotectant medium containing 10% dimethylsulfoxide (DMSO). The DMSO acts to prevent ice crystal formation during the slow freezing process, maintaining cell viability. Some cell types such as PBMC may be stored short term (less than one week) at -800C or long term in a liquid nitrogen freezer. For maximum recovery and viability upon thawing, cells should be healthy prior to freezing. Successful cell recovery from the frozen state requires rapid but gentle thawing of the cells followed by immediate removal of cryoprotectant medium from the cells. Note the protocol suggesting thawing only 1-2 vials at a time and initial 1-2 drop addition of buffer to avoid shocking the cells.
Materials

  1. Beckman Allegra X-14 with SX4750A swinging bucket rotor and inserts with biosafety covers
  2. Hemacytometer
  3. 0.04% Trypan blue exclusion dye in HBSS
  4. Biological Safety Cabinet, Class A2
  5. Household bleach (5% sodium hypochlorite) for preparation of a 10% bleach water solution
  6. Absorbent towels
  7. Sterile serological pipets 10, 5, 2,&1 ml size
  8. Pipet aide for serological pipets
  9. Sterile pipet tips
  10. Single channel air displacement pipets 5-50 uL
  11. Waste pan
  12. Sterile 15 ml conical polypropylene tubes
  13. Ice bath for tubes
  14. Racks for cryovials and for 15 ml tubes
  15. Freezing medium (10% DMSO and 90% FBS)
  16. Dimethylsulfoxide (reagent grade) Fisher BP231-1 or equivalent
  17. Fetal Bovine Serum (FBS)(screened for low cell cytotoxicity and of quality to support cell growth with low endotoxin)
  18. RPMI 1640 medium or equivalent with 8% FBS
  19. Nalgene/Nunc cryogenic vials, 1.5 ml capacity Catalog # 5000-1020
  20. Mr. Frosty, Nalgene Cryo 10 C Freezing container Cat# 5100-0001
  21. Isopropyl alcohol
  22. 0.2 μM sterile filter with sterile plastic bottle (150 to 500 ml)
  23. Refrigerated centrifuge with appropriate rotors (Beckman allegra or equivalent) same as 1st item
  24. -800freezer and liquid nitrogen freezer for long term storage

Reagent Preparation:
Cryprotectant/ Freezing medium (90% FBS/10% DMSO): Sterile filter FBS using a 0.2 micron filter with vacuum into a sterile container to remove any particulate matter. Add 10 ml DMSO per 90 ml FBS using a sterile disposable pipet. Be careful not to introduce the pipet beyond the tip to ensure ink on the serological pipet does not enter the DMSO. Do not re-filter after addition of the DMSO. Store the medium at 40C. Always use sterile technique when entering the solution container. Place the cryoprotectant medium container on ice while using to freeze cells.

For Thawing:
*** FACS media = 1% BSA in DPBS w/v (GB); if cells going into cell culture using Enzyme neutralization buffer = 10% FBS in DPBS (RM)
Freezing container: A Mr Frosty with isopropyl alcohol to the fill line is used to control the rate of cell freezing in the -800C freezer. Mr Frosty containers are cleaned and new alcohol added after every 5 uses. Mr Frosty is stored at 40C prior to freezing cells to ensure that cells do not warm as they are placed into the -800C freezer.
Safety warnings
Safety Considerations
1. All work is performed using BSL 2 procedures and following universal precautions for handling human blood and body fluids.
2. Wear thermal gloves to prevent freezer burn while handling frozen cells inside minus 80 freezer and liquid nitrogen tanks.
3. Proper balancing of centrifuge while spinning the cells is requred.
Procedure for Freezing
Procedure for Freezing
After Centrifugation at 800Xg for 10min at 40C pour off supernatant and resuspend cell pellet by gently tapping the tube by hand (with fingers) before adding the appropriate amount of freezing medium to tube to yield a cell concentration/vial as indicated in the following table. Immediately pipet 1 ml of cells in cryoprotectant freezing medium into labeled cryovials, close caps tightly and place into a Mr.Frosty slow-cooling freezing container taht was kept at room temperature.
Place the freezing container with the vials in a -800C freezer and let freeze for at least 24 hours before transferring to liquid nitrogen freezer unit for long term storage.
Follow the table to determine the number of cells to be frozen per vial
Total Number of Cells Recovered (106) # vials to freeze PBMC Concentration of Cells per Vial (106) Dissociated Organ Cells
<8 1 All Available All Available
8-10 2 Split cells between 2 vialsSplit cells between 2 vials
>10-50 2-10 ** 5 x106 cells per vial** 10 x106 cells per vial
>50 10 Divide cells evenly between 10 vials** 10 x106 cells per vial
** Round cells to the nearest vial.(ex. 21(106) PBMC cells equals 4 vials at 5.2(106)/vial)
Procedure for Thawing
Procedure for Thawing
Move tubes quickly to dry ice from liquid nitrogen freezer for transporting to laboratory. Use protective freezer gloves and a face shield when removing tubes from the liquid nitrogen freezer.
Thaw only 1-2 tubes at a time in 37o C water bath while shaking continuously just until the last ice crystal remains. This should take 65-70 seconds for vials containing 1 ml of freezingmedium.
Using a one ml pipet, transfer the entire contents from the thawed tube to a15ml conical polypropylene tube at ambient room temperature. Slowly add 5ml of cold** FACS*** media 1-2 drops at a time, while mixing carefully and thoroughly by swirling tube for the first 5 ml. Repeat for the second vial (if applicable). Add an additional 5 ml of warm FACS media to each tube before proceeding directly to next step.
After transfering the cells in freezing medium from freezing vials with 1 ml pipette, wash the empty freezing vial with 1 ml thawing medium, mix slowly to collect any leftover cells, add this to the thawed cells.
** 6/10/19 this is a change in the SOP from “warm = 25 to 370C”; however we have been using the cold buffer for some time already

*** "FACS media" = 1% BSA in DPBS w/v (GB); or if cells going into cell culture suggest using "enzyme neutralization buffer" = 10% FBS in DPBS (RM)
Centrifuge tubes at 1000 X g for 10 min at 40C remove supernatant, gently tap tube to resuspend cell pellet and add 10 ml warm FACS media. Centrifuge the tube again as above. Discard supernatant.
Resuspend to desired volume in medium.
While counting the cells under microscope, store the cells on ice.
Dilute an aliquot of cell suspension in trypan blue exclusion dye for counting in a hemocytometer to determine recovery and viability.
Analysis of Results
Analysis of Results
Determine viability and revised cell number of thawed cell aliquots by trypan blue or live/dead flow cytometry assay