EPC Derivation from Whole Blood

Transcription

1 Page 1 Overview This protocol describes procedures for isolation and expansion of human blood-outgrowth endothelial progenitor cells (EPCs) from human blood using a Ficoll gradient separation to isolate mononuclear cells (MNCs) which are then cultured in an endothelial specific medium 1. The resulting EPCs are sufficient for reprogramming using the Stemgent StemRNA -SR Reprogramming Kit (Cat. No ). This protocol outlines the procedure for isolating EPCs from 50 ml of human peripheral blood. Reagent volumes can be scaled accordingly for other blood volumes. Caution This procedure uses human blood. Human blood is a potential source of infection with blood-borne pathogens. Prior to beginning this experiment, please consult your institutional biosafety group for specific guidelines on how to minimize your possible exposure. Appropriate personal protective equipment (lab coats, gloves, safety glasses, etc.) should be worn throughout these procedures. Contents: Required Materials Experimental Timeline EPC Derivation Protocol Step 1: Material Preparation Step 2: Ficoll Separation Step 3: Derivation and Expansion of Primary EPCs Step 4: Passaging and/or Freezing of EPCs FAQs References Technical & Customer Support Trademarks Ordering Information Abbreviations DMSO EGM EPCs FBS MC mesc MNC PBS PMC srrna Dimethyl sulfoxide Endothelial growth medium Human blood-outgrowth endothelial progenitor cells Fetal bovine serum Medium change Mouse embryonic stem cell Mononuclear cell Phosphate-buffered saline Partial medium change Self-replicative RNA

2 Page 2 Required Materials PRODUCT DESCRIPTION Human donor blood (50 ml) Stored at room temperature and optimally used the day of collection Ficoll-Paque TM Premium (Ficoll) PBS (Ca, Mg-free) EGM TM -2 BulletKit TM FBS, mesc qualified Nalgene TM Rapid-Flow Sterile Disposable Filter Units 500 ml; Pore Size: 0.20 µm Rat tail collagen in 0.02 M acetic acid Acetic acid Trypsin-EDTA (0.05%) Dimethyl Sulfoxide (DMSO) CAT. NO.* GE Healthcare Life Technologies Inc. Gibco Lonza CC-3162 GE Healthcare Hyclone SH E Thermo Scientific Corning Sigma Life Technologies Sigma D2650 *Store all required materials per manufacturer s instructions.

3 Page 3 Experimental Timeline FIGURE 1. Timeline for typical experiment. Exact timing for derivation and expansion steps may vary by a few days depending on growth characteristics of the resulting EPC culture. Day 1: Prepare collagen-coated plates, isolate MNCs and culture in EPC Derivation Medium Day 3: Partial medium change (PMC): Exchange 50% of medium with fresh EPC Derivation Medium Day 4, 6, 8, 10, etc.: Medium change (MC) in EPC Derivation Medium Days 8-14: EPC colonies appear Days 14-18: Passage EPC colonies and expand in EPC Reprogramming Medium Day 16, 18, 20, etc.: MC in EPC Reprogramming Medium Days 18-25: Replate for reprogramming in EPC Reprogramming Medium

4 Page 4 EPC Derivation Protocol Step 1: Material Preparation Material preparation should be completed prior to the arrival of the blood sample. All steps should be performed under sterile conditions in a biological safety cabinet Coating T75 flask for MNC Plating 1. Dilute rat tail collagen to 50 μg/ml in 0.02 M acetic acid. 2. Use 8 ml collagen solution to cover the bottom of each of two T75 tissue culture flasks. Incubate at room temperature for one hour. 3. Aspirate collagen solution and rinse with PBS. 4. Collagen-coated flasks may be used immediately or air-dried and stored at 4 C for up to one week. NOTE: Adjust flask size for plating more or fewer cells (e.g. T25 for up to 4x10 7 cells or use two T75 flasks and split MNC suspension equally for more than 1x10 8 cells) Preparation of EPC Derivation Medium NOTE: 200 ml of EPC Derivation Medium is sufficient to culture up to 1x10 8 MNCs on a T75 flask for 20 days. 1. Thaw EBM-2 basal medium and mesc-qualified FBS in refrigerator overnight. 2. To 500 ml EBM-2 basal medium, add 100 ml mesc-qualified FBS and the EGM-2 SingleQuots (except supplied FBS) to make EPC Derivation Medium. Filter medium using 0.2 μm filter unit. NOTE: Do not use the FBS supplied in the SingleQuot kit. 3. Store EPC Derivation Medium at 4 C for up to four weeks.

5 Page 5 Step 2: Ficoll Gradient Separation MNCs are isolated from fresh human peripheral blood or cord blood by differential centrifugation. 2.1 Ficoll Centrifugation to Isolate MNCs DAY 1 For best results, blood samples should be shipped at room temperature, maintained at room temperature after arrival, and used the day of receipt. NOTE: Never shake the blood. 1. Warm Ficoll to room temperature for at least one hour. 2. Dilute 50 ml blood with 90 ml PBS. 3. Mix Ficoll stock solution by inverting several times. 4. Prepare four 50 ml conical tubes by adding 15 ml Ficoll to each tube. 5. Carefully layer 35 ml PBS-diluted blood onto the Ficoll layer in each 50 ml tube by adding the blood very slowly down the side without disturbing the Ficoll layer. 6. Centrifuge at 500 x g for 30 minutes at 18 C with the brake function on the centrifuge disabled for this step. 7. Carefully aspirate the upper layer, leaving the middle MNC layer (See Figure 2). 8. Collect each MNC layer into a separate new 50 ml conical tube. 9. Dilute each MNC layer in 3-4 volumes of PBS and centrifuge at 500 x g for 10 minutes at 18 C. 10. Combine the resulting MNC pellets, suspend the cells in 50 ml PBS, and centrifuge for 15 minutes at 200 x g. 11. Combine the resulting MNC pellets by resuspending the cells in 10 ml EPC Derivation Medium. 12. Determine an accurate cell count. 13. Proceed immediately to Step 3: Derivation and Expansion of EPCs. FIGURE 2. Ficoll gradient separation. Illustration showing 4 post-centrification layers.

6 Page 6 Step 3: Derivation and Expansion of Primary EPCs Primary EPCs are cultured from suspended MNCs on collagen-coated flasks using EPC Derivation Medium. The length of the EPC derivation process varies by donor sample. In general, the EPC colonies are ready for expansion between days 14 and 18 of the EPC derivation process. 3.1 Derivation of EPCs DAY 1 to DAY 14 NOTE: For some EPC lines, this step may require up to 18 days. Please adjust subsequent protocol times accordingly. 1. Day 1: Plate MNC suspension at a maximum density of 1.5x10 6 cells/cm 2 onto collagen-coated T75 tissue culture flask (prepared in Step 1, 1.1) equivalent to up to 1x10 8 cells per T75 using 20 ml EPC Derivation Medium per T75 flask. NOTE: Adjust flask size for plating more or fewer cells (e.g., use a T25 for up to 4x10 7 cells, or for more than 1x10 8 cells use two T75 flasks and split MNC suspension equally). 2. Culture at 37 C in incubator in 5% CO Day 3: PMC-carefully remove 10 ml of culture medium from each T75 flask and exchange it with 10 ml of fresh EPC Derivation Medium 4. Day 4, 6, 10, etc. (every other day): MC - Remove the entire volume of culture medium and replace with 15 ml of fresh EPC Derivation Medium (See Figure 3). 5. Between Day 8 and Day 14 EPC colonies should appear. Continue EPC Derivation Medium changes every other day until colonies get denser. Expect 1-12 EPC colonies per T75 flask to appear. 6. Once the EPC colonies reach the appropriate density and adopt a three-dimensional morphology they are ready to be expanded, typically between Day 14 and Day 18. NOTE: Do not let the colonies get too dense or they will start to vascularize (See Figure 4c). 10x Day 2 p0 10x Day 7 p0 4x Day 10 p0 10x Day 17 p1 10x Day 21 p1 FIGURE 3. Derivation timeline for establishment of adherent EPCs derived from human blood. A minimum of 1x10 7 human MNCs were seeded onto collagen-coated flask. Day 2 Passage 0 (p0) suspension culture of human MNCs. Day 7 EPC colonies are emerging. Day 10 EPC colony maturation Day 17 Passage 1 (p1) EPC culture 3 days after passaging. Day 21 Confluent p1 EPC culture ready to be passaged.

7 Page 7 A B Good 3-dimensional density 4x C Good 3-dimensional density 4x Too dense (vascularized) 4x FIGURE 4. Colony morphology. Examples of EPC colonies ready to be passaged to p1 (A,B) and vascularized EPC colonies (C) that have grown too dense. 3.2 Preparation of EPC Reprogramming Medium NOTE: EPC Reprogramming Medium is used for expansion, late passaging and reprogramming. 50 ml of the EPC Reprogramming Medium is sufficient to reprogram one well of a 6-well plate. 1. Thaw mesc-qualified FBS in refrigerator overnight 2. Add 50 ml mesc-qualified FBS and the EGM-2 SingleQuots (all except for SingleQuot-supplied heparin and FBS) to 500 ml EBM-2 basal medium to make EPC Reprogramming Medium. Filter medium using 0.2 μm filter unit. NOTE: Do not add heparin or FBS from the SingleQuots kit to make EPC Reprogramming Medium. Heparin is a highly charged protein that will interfere with transfection reagents. 3. Store EPC Reprogramming Medium at 4 C for up to four weeks. 3.3 Passaging and Expansion of Primary EPC Culture - DAY 14 NOTE: Passaging occurs at Day 14; expansion typically lasts another 4-7 days. 1. Remove the culture medium from the original T75 derivation flask of p0 cells to be harvested. 2. Add 10 ml of PBS to the culture surface of the flask to wash. Aspirate the PBS. 3. Add 3 ml of 0.05% Trypsin/EDTA to the culture surface of the flask and incubate for 3 to 5 minutes at 37 C and 5% CO Tap the flask until the EPC colonies are completely detached from the culture surface. 5. Add 6 ml of EPC Reprogramming Medium to the flask to neutralize the Trypsin/EDTA. 6. With a 5 ml pipet, gently pipette the suspended cells up and down until the colonies dissociate into single cells. Transfer the harvested EPC cell suspension from the T75 flask to a 15 ml conical tube.

8 Page 8 7. Centrifuge the cells for 5 minutes at 250 x g. 8. Remove the supernatant and resuspend the pellet in 5 ml of EPC Reprogramming Medium. 9. Plate EPCs in a T25 tissue culture flask without collagen coating to seed a p1 culture. NOTE: If an insufficient number of cells are obtained (only one small colony or very slow growing colonies), suspend the cells in 2 ml EPC Reprogramming Medium and plate in one well of an uncoated 6-well tissue culture plate (see Figure 5). 10. Replace the medium every other day with fresh EPC Reprogramming Medium. 11. When p1 EPCs reach 80% -90% confluence (typically about 4-7 days (see Figure 6)), passage and plate cells for reprogramming according to protocol: StemRNA-SR Reprogramming Using Blood-Derived EPCs Day 18 p0 4x FIGURE 5. Insufficient cells. Example of an insufficient number of cells in a p0 EPC colony on Day 18 that should be plated for expansion into one well of a 6-well plate. Day 24 p1 4x FIGURE 6: p1 EPCs ready for re-plating and reprogramming. These cells are ready for re-plating into a 6-well plate for a StemRNA-SR reprogramming experiment or replating for further expansion (see Step 4). NOTE: Stemgent recommends proceeding directly to EPC reprogramming. After plating EPCs for reprogramming, unused cells can be frozen down at 5x10 5 cells/vial (see step 4) or further expanded. Step 4: Passaging and/or Freezing of EPCs Unused EPCs can be frozen or passaged further for later use. Generally, the reprogramming efficiency of cryopreserved EPCs is lower compared to using fresh EPCs, and EPC reprogramming efficiency decreases with higher passage number. 1. For Cryopreservation: After trypsinization, spin down EPCs at 500 x g for 5 minutes and resuspend 5.0x10 5 EPCs in 500 µl of 90% FBS/10% DMSO. Store cryovial in isopropanol cooling chamber overnight at -80 C and transfer the next day into liquid nitrogen tank. 2. For Passaging, passage 5x10 5 cells in a T25 flask without collagen coating.

9 INTEGRATED TOOLS FOR TRANSLATIONAL RESEARCH FAQs Q: Can I start with frozen MNCs from e.g., cord blood and peripheral blood? A: While one can culture EPCs from frozen MNCs, generally this gives fewer EPC colonies. We recommend starting with fresh blood if available. If only frozen MNCs are available, thaw 5 x 10 7 cells. Wash with PBS, then resuspend in EPC Derivation Medium. Proceed to Step 3 of this protocol. References 1. Geti, et al. A Practical and Efficient Cellular Substrate for the Generation of Induced Pluripotent Stem Cells from Adults: Blood Derived Endothelial Progenitor Cells. Stem Cell Trans Med 1:855 (2012) Technical and Customer Support To obtain the latest technical or customer support information, please visit where you have access to the following information: Telephone and fax information for order placement and technical support Product specification sheets, protocols, application notes and other product support documentation Stemgent protocol video gallery Information on customer training and workshops Technical Support tech.support@stemgent.com Customer Service orders@stemgent.com Trademarks Ficoll-Paque is a trademark of GE Healthcare. EGM, BulletKit and SingleQuots are trademarks of Lonza. Nalgene and Rapid-Flow are registered trademarks of Thermo Scientific. Unless otherwise noted, ReproCELL, Inc. and ReproCELL, Inc. logo, BioServe Biotechnologies Ltd. and BioServe Biotechnologies Ltd. logo, Stemgent, Inc. and Stemgent, Inc. logo, Reinnervate Ltd., and Reinnervate Ltd. logo and all other trademarks are the property of ReproCELL Inc ReproCELL, Inc. All rights reserved. Ordering Information For the latest products and services available through Stemgent A ReproCELL Group Company, please visit our website(s) or inquire via , phone or fax provided below. AMERICAS-HUMAN TISSUE 9000 Virginia Manor Road, Suite 207 Beltsville, MD 20705, USA Tel: (301) info@bioserve.com AMERICAS-REAGENTS 51 Moulton Street Cambridge, MA 02138, USA Tel: (617) info@stemgent.com ASIA PACIFIC KDX Shin-yokohama381 Bldg9F , Shin-yokohama, Kohoku-ku, Yokohama, Kanagawa , Japan Tel: info_jp@reprocell.com EUROPE NETPark Incubator Thomas Wright Way Sedgefield, Co. Durham TS21 3FD, UNITED KINGDOM Tel: +44 (0) info@reinnervate.com