Important Note: FH and media must be at room temperature (20-24 C) for accurate gradient formation

Transcription

1 Procedure: Lymphocyte Isolation Using a Density Gradient Materials: Method: Procedure: Lymphocyte isolation using density gradient centrifugation allows the separation of lymphocytes for crossmatch testing from red blood cells, granulocytes, dead cells, etc. Density gradient centrifugation separates cells by their specific gravity. Lymphocytes, which have a specific gravity of 1.070, will collect at the interface between the suspension medium and a ficoll-hypaque (FH) solution that has a specific gravity of It is estimated that each 10 ml ACD tube will yield 1 NUNC with 5-10 million cells. Important Note: FH and media must be at room temperature (20-24 C) for accurate gradient formation Ficoll-Hypaque Solution (FH) specific gravity: OR use CPT tubes containing density gradient (follow package instructions for use) Phosphate Buffered Saline (PBS) RPMI Freezing and Thawing (F&T20) medium o Complete Medium RPMI 1640 L-glutamine (final conc: 2mM) HEPES (ph 7.4, final conc: 25mM) Gentamycin (final conc: 50 mg/l) 20% Fetal Calf Serum (FCS) or Fetal Bovine Serum (FBS) heatinactivated (1 hr at 56 C) Filtered via 0.22µm filter assembly Table Top or Beckman Centrifuge Conical tube (50mL) Electronic pipette (if available) Disposable Pipettes 1. Whole blood drawn in ACD tubes, spin tubes at 700 g for 5 minutes in a centrifuge. 2. Harvest the buffy coat, going slightly into the red blood cell layer. Transfer the buffy coat from 2-3 blood tubes into a labeled 50 ml conical tube. 3. Dilute to a volume of ~37ml with room temperature RPMI or PBS, cap, and invert to mix. 4. Fill a 10ml serologic pipette with 10mLs of FH solution. Layer it under the sample, by placing the full pipette into the test tube and SLOWLY dispense onto the bottom of the tube. Take caution to maintain the interface between the FH and the blood. 5. Spin tube at g for 20 minutes in a centrifuge. Page 1 of 2

2 Procedure: Lymphocyte Isolation Using a Density Gradient Note: FH is toxic to cells so exposure should be limited and at least two washes performed after gradient isolation. 6. Gently harvest the lymphocyte layer from FH interface with pipette, transfer to another labeled 50ml conical tube and add RPMI or PBS to a volume of 50 ml for a second wash. Note: Avoid collecting FH when harvesting lymphocyte layer. Carryover FH must be adequately diluted to allow pelleting of the cells during the wash steps. 7. Spin the test tube in the centrifuge for 10 minutes at 500 to 700g to pellet the cells in the bottom of the test tube. 8. Pour off the supernatant to discard, combine cell pellets if needed. Repeat wash for a total of 2 washes. 9. Resuspend cell pellet in 2mL F&T20. Break up the cell pellet by gentle mixing. 10. Check lymphocyte viability and cell count. References: ASHI Laboratory Manual, 3rd Edition, 1994, I.A Tissue Typing Reference Manual (SEOPF), 1993, B Page 2 of 2

3 Procedure: Freezing Bulk Lymphocytes Purpose: Materials: This procedure describes how to store lymphocytes long term, in a frozen viable state for future testing. The condition of the cells at the time of freezing is critical to future recovery and viability; cells must be healthy with good viability when frozen. For ACD blood, cells should be isolated and frozen within 2 days of phlebotomy. Care must be taken when freezing cells. The formation of ice crystals in and around the cells in suspension can fracture cell membranes, causing cells to lose their integrity. For bulk cell preservation, a gradual cooling of the cells at a rate of 1 o C/minute is optimal. DMSO increases membrane permeability and, in turn, protects the cells during freezing by reducing the formation of intracellular ice and reducing solute concentrations thus reducing ionic stress to the cells. However, DMSO is toxic to cells at high concentration at room temperature. Further, addition of DMSO to aqueous solutions is an exothermic reaction, resulting in heat generation, which can be damaging to cells. Therefore, undiluted DMSO must never be added directly to a cell suspension and diluted DMSO must be added dropwise. Cells must be kept in an ice bath while exposed to DMSO. The loading of cells into storage units and the transfer of cells from the ice bath into the -80 C (+/- 5 C) freezer and later to liquid nitrogen should be rapid, to prevent any warming of the cell suspension/freezing mixture. Freezing and Thawing (F&T20) medium o Complete Medium: RPMI 1640 L-glutamine (final conc: 2mM) HEPES (ph 7.4, final conc: 25mM) Gentamycin (final conc: 50 mg/l) 20% Fetal Calf Serum or Fetal Bovine Serum (FCS/FBS) heatinactivated (1 hr at 56 C) Filtered via 0.22µm filter assembly 15-20% DMSO in F&T20 medium Pipettes NUNC Cryovials Centrifuge Ice bucket Test tubes Controlled Rate Freezer OR -80 C freezer Liquid nitrogen freezers Page 1 of 2

4 Procedure: Freezing Bulk Lymphocytes Method: 1. Isolate lymphocytes using a density gradient procedure a. Viability must be within acceptable range >80%. 2. Adjust cell concentration to 1-2 x 10 7 cells/ml using cold F&T20. Cool the cell suspension in an ice bath. 3. Keep the cell suspension in an ice slush during this step. Add an equal volume of chilled 15% DMSO solution drop-wise with constant mixing. 15% is only a guideline. 4. Transfer 1ml into a labeled, chilled cryogenic vial on ice. (0.5-1 x 10 7 cells/vial) a. Manual Method: -80 C freezer: i. Place the vials in a -80 C (+/- 5 C) freezer. Perform this transfer immediately. Cells must remain in the -80 C (+/- 5 C) freezer for at least 4 hours but no more than 3 days. Vials must be placed in a location that is not subject to temperature fluctuations when the freezer is opened. Alcohol chamber (i.e. Mr. Frosty ) may be used to slow the rate of freezing and sustain viability. ii. Transfer the frozen cell preparations for long-term storage into vapor phase of liquid nitrogen. b. Automated Rate Freezer Method: i. Follow manufacturer s instructions for rate freezer. Liquid nitrogen valves and rate freezer should be turned on to cool chamber prior to depositing the cryogenic vials. Freezing cycle is completed in approximately 1 hour. ii. Transfer the frozen cell preparations immediately for long-term storage into vapor phase of liquid nitrogen. References: ASHI Laboratory Manual, 4th Edition, 2000 pgs. II.A Current Protocols In Immunology, 1997 A.3.G.1-3. Page 2 of 2

5 Procedure: Thawing Bulk Lymphocytes Purpose: Multiple factors impact cell viability when thawing cryo-preserved cells. These include: (1) Ion gradients; high ion concentrations will thaw first. Gentle mixing upon thawing assures homogeneity and reduces ion gradient formation. (2) Intracellular ice crystals, cells should be thawed rapidly in a 37 o C (+/- 5 o C) water bath with constant agitation to prevent the formation of intracellular ice, which can puncture cell membranes. (3) DMSO toxicity, washing to remove DMSO should occur at the earliest possible time. (4) Osmotic swelling, the wash media should be added slowly to reduce swelling upon rehydration of the cells. (5) Cells that have been frozen and thawed are fragile and can be ruptured by the force generated by high speed centrifugation and vortexing. This can be avoided by centrifuging and vortexing the cells at a low speed for a longer time. (6) Long exposure to Ficoll-Hypaque is toxic to cells and gradient centrifugation should only be performed when absolutely necessary. Materials: Freezing and Thawing (F&T20) medium: o Complete Medium RPMI 1640 L-glutamine (final conc: 2mM) HEPES (ph 7.4, final conc: 25mM) Gentamycin (final conc: 50 mg/l) 20% Fetal Calf Serum or Fetal Bovine Serum (FCS/FBS) heat-inactivated (1 hr at 56 C) Filtered via 0.22µm filter assembly RPMI or PBS media with 10% FCS (or alternative protein source) Pipettes Frozen vial of lymphocytes 15mL test tubes Water bath Method: 1. Remove desired vials from liquid nitrogen storage and place immediately in a 37 C (+/- 5 C) water bath, gently swirling the samples in the water bath to mix. 2. As the last ice crystal is about to melt, remove the cap. Add F&T20 dropwise to the cells, mixing continuously until the freezing vial is full. 3. Transfer the contents of the vial to a centrifuge tube and begin adding the F&T20 slowly (10mL over a 2 minute time interval) with constant, gentle mixing. Page 1 of 2

6 Procedure: Thawing Bulk Lymphocytes 4. Centrifuge for 10 minutes at 500xg to pellet the lymphocytes and discard the supernatant. 5. Resuspend the lymphocytes with wash buffer and mix thoroughly. Pellet the cells by centrifugation at 500xg for 10 minutes. 6. Resuspend the cells in appropriate media and check the viability of the lymphocytes. Note: Viability of thawed cells should be >80%. If not, ficoll-hypaque gradient separation may be performed to remove dead lymphocytes. References: ASHI Laboratory Manual, 4th Edition, 2000 pgs. II.A Page 2 of 2

7 Cryo Packaging and Shipping Instructions Last Revised 2/10/15 1. General a. Cryo kits requested by 14:00 local time must be shipped by the donor center on the same day for overnight AM delivery using FEDEX or UPS. b. Cryo kits requested after 14:00 local time must be shipped by the donor center on the next business day for overnight AM delivery using FEDEX or UPS. c. Cryo Kits should not be shipped the day before a weekend (i.e. Friday) or the day before a holiday. d. To view Illustrations related to these instructions visit the NKR Website at 2. Packaging a. Remove the lid of the Styrofoam container then remove contents and set aside. b. Cover the bottom 1/3 of the Styrofoam container with dry ice. c. Place the NUC into the cryovial box then close and place the box on the dry ice. d. Ensure the cryovial box is fully surrounded by dry ice. e. Place the lid on the Styrofoam container. f. Place the Styrofoam container into the outer shipping box and seal the box. 3. Class 9 label a. Grab the class 9 label that you set aside earlier. b. Fill in the weight of the dry ice (Generally 5 lbs) in the space provided on the Class 9 label if it is not pre-filled. c. Fill out the shipper name and address (your center/lab) on the Class 9 label. d. Fill out the consignee name and address (recipient center/lab) on the Class 9 label using the receiving center/lab information provided in the requesting the NUC. e. Affix the Class 9 Label to the outer shipping box. 4. Air bill a. Get a shipping air bill from your mailroom or shipping vendor. b. Fill out the air bill including the name and telephone number of the consignee provided in the requesting the NUC.