TITLE: 703.3 URMC HTC_Cryopreservation_of_Isolated_Cells_061220
AUTHORS: Gloria Pryhuber, Ravi Misra, Heidie Huyck, Gautam Bandyopadhyay

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Lung MAP HTC - BioRepository for Investigation of Neonatal Diseases of the Lung (BRINDL)</span></div></div><div class = "text-block"><span style = "font-weight:bold;">Standard Operating Procedures</span><span>            </span><span style = "font-weight:bold;">703.2_HTC102314rev061019</span></div><div class = "text-block"><span style = "font-weight:bold;">Cryopreservation of Isolated Cells</span></div><div class = "text-block">Purpose and Scope of the Procedure or Laboratory Assay</div><div class = "text-block"><ol style = "list-style-type: decimal;"></ol></div></div>

[STEPS]
?. [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.
?. [Procedure for Freezing]
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.
?. [Procedure for Freezing]
Follow the table to determine the number of cells to be frozen per vial  ABCD1Total Number of Cells Recovered
 (106)  # vials to freeze
    PBMC
  Concentration of Cells per Vial (106)
    Dissociated 
  Organ Cells
  2  &lt;8
    1
    All Available
    All Available
  3  8-10
    2
  Split cells between 2 vialsSplit cells between 2 vials4  &gt;10-50
    2-10
  ** 5 x106 cells per vial** 10 x106 cells per vial5  &gt;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)
ABCD1Total Number of Cells Recovered
 (106)  # vials to freeze
    PBMC
  Concentration of Cells per Vial (106)
    Dissociated 
  Organ Cells
  2  &lt;8
    1
    All Available
    All Available
  3  8-10
    2
  Split cells between 2 vialsSplit cells between 2 vials4  &gt;10-50
    2-10
  ** 5 x106 cells per vial** 10 x106 cells per vial5  &gt;50
    10
  Divide cells evenly between 10 vials** 10 x106 cells per vial
?. [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.
?. [Procedure for Thawing]
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.
?. [Procedure for Thawing]
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.
?. [Procedure for Thawing]
** 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
?. [Procedure for Thawing]
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.
?. [Procedure for Thawing]
Resuspend to desired volume in medium.
?. [Analysis of Results]
Determine viability and revised cell number of thawed cell aliquots by trypan blue or live/dead flow cytometry assay
?. [Procedure for Thawing]
*** "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)
?. [Procedure for Thawing]
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.
?. [Procedure for Thawing]
While counting the cells under microscope, store the cells on ice.
?. [Procedure for Thawing]
Dilute an aliquot of cell suspension in trypan blue exclusion dye for counting in a hemocytometer to determine recovery and viability.