Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

Biology

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Summary

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

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Hofmann, N. A., Reinisch, A., Strunk, D. Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells. J. Vis. Exp. (32), e1524, doi:10.3791/1524 (2009).

Abstract

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion >1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4

This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Protocol

A.) ECFC ISOLATON

DAY 1:

  1. Before you start with the experiment prepare the cell culture medium Endothelial Growth Medium-2 (EGM-2). Supplement 500ml Endothelial Basal Medium-2 with heparin (1ml of 1000 U/ml), 5ml 100x solution of penicillin/ streptomycin (all Sigma, St. Louis, MO; www.sigmaaldrich.com), L-glutamine (2 mM final concentration), and 50 ml pooled human platelet lysate (pHPL). Then add 0.2 ml hydrocortisone, 2 ml hFGF-B, 0.5 ml VEGF, 0.5 ml EGF, 0.5 ml IGF-1 and 0.5 ml ascorbic acid (provided as SingleQuots, all Lonza, Walkersville, MD; http://www.lonzabioscience.com/). Sterile filtrate the medium through a 0.2 m-pore size 500 ml Millipore vacuum filter. Since pHPL forms small clots you may need two filters for one medium. Pre-warm filtered EGM-2 to 37°C in a water bath
  2. Collect the blood samples by drawing 5 ml of adult human peripheral blood from the cubital vein into a 6ml preservative free sodium-heparin blood vacutainer tube. You should process the blood samples within a maximum of two hours.
  3. Start by preparing one 75 cm2 (T75; Costar) flask with a vented cap, one 25 ml pipette and two 5 ml pipettes in a laminar flow bench.
  4. Pre-fill 15 ml pre-warmed supplemented EGM-2 into the flask with the 25 ml pipette. Now open the blood vial and sample all 5 ml with the 5 ml pipette into the flask. Rinse the empty blood vial with 5 ml of additional EGM-2 with the second 5 ml pipette. The total volume within the T75 is 25 ml. Close the vented-cap of the flask and place in an incubator at 37°C, 5 % CO2 and 95 % humidity for 24 hours.

DAY 2:

  1. After approximately 24 hours (over night) you should remove the medium-blood mixture to get rid of non adherent cells. For this prepare two 25 ml pipettes and three 5 ml pipettes in the laminar flow, pre-warm 20 ml of supplemented EGM-2 (prepared on the previous day) and 30 ml PBS to 37°C in a water bath.
  2. Remove the flask with the sample from the incubator and remove all the supernatant with a 25 ml pipette. Take care not to scratch the flask surface to avoid damaging any potential colony. Wash the inner flask cell adherence surface three times by gently adding 10 ml of pre-warmed PBS and tilting the flask from one side to the other. Remove the PBS immediately each time and discard.
  3. Add 20 ml of fresh pre-warmed EGM-2 to the flask and place it back into the incubator. This process should be performed as fast and gentle as possible for the attached cells not to detach. ECFCs detach easily if they are kept in PBS or at room temperature for to long.
  4. Screen through the flask systemically every second day starting at day two using a low magnification of a light microscope looking for ECFC outgrowth. When a colony is found mark on top at the outer side of the flask indicating the position of the colony. Note that the flaks should not be outside of the incubator for more than 5 minutes.

DAY 5:

  1. On the fifth day after seeding perform a complete medium change to remove non-adherent cells. For this purpose pre-warm 20 ml of EGM-2 to 37°C in a water bath and prepare two 25 ml pipettes.
  2. Remove the complete medium using a 25 ml pipette and add fresh, pre-warmed supplemented EGM-2 as soon as possible. Cells should not be left without medium for more than a minute to avoid drying. Place back into the incubator and repeat screening the flask daily.
  3. Feed the cells by replacing 30 % of medium by fresh supplemented EGM-2 twice weekly.

DAY 14-28:

  1. Typical colonies with cobble stone morphology should appear around day 12. Mark the top outer side of the flask to indicate the position of each colony. Allow the colonies to grow until day 28 unless single cells start to detach. Once, primary colonies have been identified they can be harvested between days 14 and 28, depending on their size.

B.) ECFC HARVEST

  1. Pre-warm 5 ml trypsin 0.05%/EDTA, 20 ml PBS and 5ml fresh supplemented EGM-2.
  2. Completely remove medium from the flask into a Falcon tube (take care not to touch the surface of the flask to avoid damaging any potential colony) and wash cells carefully with 10 ml preheated PBS.
  3. Add 5 ml trypsin/EDTA and incubate for 5 minutes in the incubator. The cells should not be exposed to trypsin for more than 7 min since it becomes toxic. Thoroughly tapping the sides of the flask helps cells to detach. Quench trypsin protease activity by adding approximately 10 ml of the preserved used culture medium.
  4. Remove cell suspension into a 50 ml Falcon tube. Wash the flask once with 10 ml PBS which is thereafter added to the harvested cell suspension. Centrifuge the cell suspension at 300 g for 7 min at 4°C to collect the cells and remove the trypsin. Discard the supernatant and resuspend the cell pellet immediately in 1 ml PBS. Keep on ice.
  5. Determine cell number as described in a JoVE protocol by R. Ricardo and K. Phelani.5

C.) ECFC EXPANSION

  1. Supplement 500 ml EGM-2 as described in A 1.1). Pre-warm to 37°C in a water bath.
  2. For approximately 2500 cm2 culture space prepare a either 11 T220 (225 cm2) or 15 T175 (175 cm2) or one four-layered cell factory (CF-4; Nunc, Naperville, IL; http://www.nuncbrand.com). Additionally you will need two fresh vent-caps and a special sterile funnel in the laminar flow bench.
  3. To seed ECFCs at a starting cell density of 100 c/cm2 calculation of the cell number is necessary. For one CF-4 252,800 cells are resuspended in 500 ml fresh EGM-2 and poured into the CF-4 using the sterile funnel. Close one vent with the vent-cap and tilt the CF-4 to one longitude side, enabling an equal distribution of the medium between all 4 flours. Then tilt the chamber back and open the lid of the vent-cap. Place CF-4 in incubator.
  4. Change 20 % of the medium at least once weekly until CF-4 is confluent.
  5. Harvest cells as described in B.) by using 70 ml of trypsin/EDTA and approximately 200 ml PBS.

REPRESENTATIVE RESULTS

Using this isolation method we observed a primary colony formation at day 12. In a previous study were able to recover a mean of 4.0 ± 0,8 ECFC-colonies derived from each ml of peripheral blood.4 ECFCs showed a typical cobble stone appearance. After large scale expansion, with a seeding density of 100 cells/cm2, we obtained > 1 x 108 cells in a total of approximately 30 days from 3 male and 1 female volunteers (age between 26 and 50 years).

ECFC cultured under these conditions were shown to be proliferative, functional and genomically stable.4 Furthermore cells can be stored (in 10 % DMSO) in liquid nitrogen for further use. 3,4

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Discussion

This novel isolation and expansion method is a simple and efficient process that is more efficient and less time consuming by avoiding any cell separation (no density gradient needed) and less expensive than conventional techniques. By using pHPL contact to bovine antigens and putative subsequent xenoimmunization is excluded. During the culture period small clots can form, caused by the pHPL. Based on our experience these clots do not effect colony formation and cell proliferation or function.

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Acknowledgments

The Authors thank Dr. Katharina Schallmoser for providing pHPL and Margaretha Frühwirth and Daniela Thaler for excellent technical assistance.

Materials

Name Company Catalog Number Comments
Endothelial basal Medium-2 Lonza Inc. 500ml
EGM-2 SingleQuots Lonza Inc. EGF, hFGF-B, VEGF, IGF-1, Hydrocortisone, ascorbic acid
preservative-free Heparin Biochrom AG 10U/ml per EGM-2
L-Glutamine Sigma-Aldrich 2mM per EGM-2
Penicillin and Streptomycin Sigma-Aldrich 100U/mL Penicillin and
100μg/mL Streptomycin per
EGM-2
Trypsin/1mM EDTA Invitrogen 7ml for T75 flask
70ml for CF-4
PBS
Four-layered cell factory (CF-4) Corning
Sterile Funnel Corning
T75cm2 culture flask (with vented cap) Corning
6 mL vacutainer tubes (preservative-free sodium heparin) BD Biosciences preloaded with 108 IU/6ml of
preservative-free sodium heparin
50ml Falcon tubes Falcon BD
5ml Stripetts Costar
25ml Stripetts Costar
Squeezer Costar
0,2 pore size sterile filter EMD Millipore 2x 500ml per EGM-2

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References

  1. Solovey, A., Lin, Y., Browne, P., Choong, S., Wayner, E., Hebbel, R. P. Circulating activated endothelial cells in sickle cell anemia. N Engl J Med. 337, 1584-1590 (1997).
  2. Yoder, M. C., Mead, L. E., Prater, D. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood. 109, 1801-1809 (2007).
  3. Schallmoser, K. Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum. Tissue Eng Part C Methods. 14, 185-196 (2008).
  4. Reinisch, A. Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo. Blood. (2009).
  5. Ricardo, R., Phelan, K. Counting and Determining the Viability of Cultured Cells. J Vis Exp. (2008).

Comments

3 Comments

  1. We tried this protocol and did not get colonies. Has anyone else gotten this protocol to work? Regards,
    Diana

    Reply
    Posted by: Diana P.
    March 26, 2010 - 12:58 PM
  2. Hi Diana,

    We obtained one colony from ² subjects out of 10 healthy donors. For each subject, we plated total ²0ml of blood in four T75 flasks. We were able to expand the cells up to 1.1 X 10E6 into one T75 flask after 48 hours of culture. Cells are currently being expanded in the cell factory. Unfortunately we were not able to reproduce the results plating blood from the same subject that we got the colony. Maybe we still need to improve our technique or maybe there are unknown variables that can affect the amount of ECFC in the circulation at the time of blood collection.

    Micheline

    Reply
    Posted by: Anonymous
    May 7, 2010 - 2:27 PM
  3. Hi Micheline
    Do you use pHPL to replace FBS? Since we followed the author's protocol, very few adherent cells were got after the wash with PBS three times.

    Reply
    Posted by: he f.
    July 19, 2011 - 8:42 AM

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