다양한 배양 조건에서 제대혈 유래 CD34+ 조혈 세포의 체외 증식

최용운; 오덕재; Yong Woon Choi; Duk Jae Oh

Korean Chemical Engineering Research, Vol.44, No.1, 73-80, February, 2006

Export Citation

다양한 배양 조건에서 제대혈 유래 CD34+ 조혈 세포의 체외 증식

Ex vivo Expansion of CD34+ Hematopoietic Cells from Cord Blood in Various Culture Environments

최용운, 오덕재^†

세종대학교 공과대학 생명공학과, 143-747 서울시 광진구 군자동 98

Yong Woon Choi, and Duk Jae Oh^†

Department of Bioscience and Biotechnology, Sejong University, 98, Gunja-dong, Gwangjin-gu, Seoul 143-747, Korea

E-mail:

초록

본 연구에서는 제대혈 유래의 조혈줄기세포를 효과적으로 배양하기 위한 선행 연구로서 세포 배양 환경에 따른 조혈줄기세포 증식능의 변화를 관찰하였다. 제대혈의 단핵구 세포에서 분리한 CD34+ 세포를 성장인자 조성-I(이하, coc-I) (EPO, GM-CSF, SCF, IL-3) 및 성장인자 조성-II(이하, coc-II) (TPO, G-CSF, SCF, IL-6, Flt3/Flk-2 ligand)가 포함되어 있는 IMDM(Iscove’s modified Dulbecco’s medium) 및 무혈청 배지(serum free media, SFM)에서 배양하였으며, 우태아혈청(FBS)의 첨가 영향, 2차원 및 3차원 배양 후 각 조건에서의 세포 증식 및 콜로니 형성능을 비교하였다. 일반적 으로 coc-I에서의 세포 증식 및 콜로니 증식이 coc-II에서보다 높았다. 3차원 배양(methocult)에서는 가장 높은 세포 증식(2,258±456배)을 나타냈으며, 같은 조성의 2차원 배양(IMDM + coc-I + FBS)에서는 가장 높은 콜로니 증식(BFU-E:652±19, CFU-GM: 520±58, CFU-GEMM: 339±100배)이 나타났다. 배지를 기준으로 보면, coc-II 조성에 우태아혈청이 포함되지 않은 경우를 제외한 모든 경우에서 세포 증식 및 콜로니 증식이 무혈청 배지에서보다 IMDM에서 높았다. 결론적으로, 모든 배양 조건 중에서 ‘IMDM + coc-I + FBS’ 및 ‘IMDM + coc-I’에서 가장 좋은 콜로니 증식을 보였으며, 우태아혈청의 첨가 및 2차원 배양 조건이 콜로니 증식에 더 효과적인 것으로 확인되었다. 본 연구 결과는 앞으로 조혈줄기세포의 체외 증식에 필요한 공정개발이나 생물반응기 설계에 유용한 정보를 제공할 수 있을 것으로 사료된다.

This study compared cell expansion and colony forming ability in human cord blood stem cells cultured exvivo with two kinds of cytokine combinations, two kinds of media, presence or absence of fetal bovine serum (FBS) and two or three dimensional (2D or 3D) culture environments. Purified CD34+ cells were cultured in the IMDM (Iscove’s Modified Dulbecco’s Medium) and SFM (Serum Free Medium) containing a cytokine cocktail.I (coc-I) (EPO, GMCSF, SCF, and IL-3) or a cytokine cocktail.II (coc-II) (TPO, G-CSF, SCF, IL-6, and Flt3/Flk-2 ligand) with or without FBS. Generally, higher cellular and clonogenic expansion were observed in the coc-I cytokine condition, compared to coc-II cytokine condition. 3D (Methocult) and 2D (IMDM + coc-I + FBS) conditions gave the greatest cell (2,258±456 fold) and CFU (BFU-E: 652±19, CFU-GM: 520±58, CFU-GEMM: 339±100 fold) expansions, respectively. In aspect of medium, IMDM was better than SFM, except for coc-II condition without FBS. In conclusion, ‘IMDM + coc-I + FBS’ and ‘IMDM + coc-I’ were the best CFU expansions on the occasion of all culture conditions. FBS and 2D conditions had affirmative effect on CFU expansion, generally. These data might provide a variety of notions about ex vivo expansion of hematopoietic stem cells.

Keywords:Ex vivo Expansion;Cord Blood;CD34+ Cell

[References]

Zon LI, Blood, 86(8), 2876 (1995)
Hann IM, Bodger MP, Hoffbrand AV, Blood, 62(1), 118 (1983)
Linch DC, Knott LJ, Rodeck CH, Huehns ER, Blood, 59(5), 976 (1982)
Gluckman E, Broxmeyer HA, Auerbach AD, Friedman HS, Douglas GW, Devergie GW, Esperou A, Thierry D, Socie G, Lehn P, N Engl J Med, 321(17), 1174 (1989)
Kogler G, Radke TF, Lefort A, Sensken S, Fischer J, Sorg RV, Wernet P, Exp. Hematol., 33(5), 573 (2005)
Ende M, Pac Med Surg, 74(2), 80 (1966)
Kubanek B, Ferrari L, Tyler WS, Howard D, Jay S, Stohlman F, Blood, 32(4), 586 (1968)
Stohlman F, Semin. Hematol., 4, 304 (1967)
Metcalf D, Science, 254(5031), 529 (1991)
Ihle JN, Chem. Immunol., 51, 65 (1992)
Leary AG, Wong GG, Clark SC, Smith AG, Ogawa M, Blood, 75(10), 1960 (1990)
Socinski MA, Cannistra SA, Elias A, Antman KH, Schnipper L, Griffin JD, Lancet, 1(8596), 1194 (1988)
Migliaccio G, Migliaccio AR, Adamson JW, Blood, 72(1), 248 (1988)
Tomonaga M, Golde DW, Gasson JC, Blood, 67(1), 31 (1986)
Clark SC, Kamen R, Science, 236(4806), 1229 (1987)
Ohara A, Suda T, Saito M, Miura Y, Okabe T, Takaku F, Exp. Hematol., 15(6), 695 (1987)
Desauvage FJ, Hass PE, Spencer SD, Malloy BE, Gurney AL, Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang WJ, Oles KJ, Hultgren B, Solberg LA, Goeddel DV, Eaton DL, Nature, 369(6481), 533 (1994)
Bartley TD, Bogenberger J, Hunt P, et al., Cell, 77(7), 1117 (1994)
Hannum C, Culpepper J, Campbell D, et al., Nature, 368(6472), 643 (1994)
Lyman SD, James L, Vanden Bos T, et al., Cell, 75(6), 1157 (1993)
Rennick D, Jackson J, Yang G, Wideman J, Lee F, Hudak S, Blood, 73(7), 1828 (1989)
Hodgkin PD, Bond MW, O'Garra A, Frank G, Lee F, Coffman RL, Zlotnik A, Howard M, J. Immunol., 141(1), 151 (1988)
Caracciolo D, Clark SC, Rovera G, Blood, 73(3), 666 (1989)
Brandt J, Briddell RA, Srour EF, Leemhuis TB, Hoffman R, Blood, 79(3), 634 (1992)
Brandt JE, Bhalla K, Hoffman R, Blood, 83(6), 1507 (1994)
Lebkowski JS, Schain LR, Okarma TB, Stem Cells, 13(6), 607 (1995)
Daley JP, Daley BM, Wysocki MG, Caligiuri MA, Biddle WC, Ex Vivo Expansion of Human Hematopoietic Progenitor Cells in Serum-free STEMPRO TM-34 Medium, pp. 62-67. vol. 18. Life Technologies, Gaithersburg, U. S. A. (1996)
Piacibello W, Sanavio F, Garetto L, Severino A, Dane A, Gammaitoni L, Aglietta M, Leukemia, 12(5), 718 (1998)
Balducci E, Azzarello G, Valenti MT, et al., Stem Cells, 21(1), 33 (2003)
Mobest D, Mertelsmann R, Henschler R, Biotechnol. Bioeng., 60(3), 341 (1998)
Poloni A, Giarratana MC, Firat H, et al., Hematol Cell Ther, 39(2), 49 (1997)
Gilmore GL, DePasquale DK, Lister J, Shadduck RK, Exp. Hematol., 28(11), 1297 (2000)
Shadduck RK, Gilmore GL, Lister J, Stem Cells, 18(2), 154 (2000)

[1] Zon LI, Blood, 86(8), 2876 (1995)

[2] Hann IM, Bodger MP, Hoffbrand AV, Blood, 62(1), 118 (1983)

[3] Linch DC, Knott LJ, Rodeck CH, Huehns ER, Blood, 59(5), 976 (1982)

[4] Gluckman E, Broxmeyer HA, Auerbach AD, Friedman HS, Douglas GW, Devergie GW, Esperou A, Thierry D, Socie G, Lehn P, N Engl J Med, 321(17), 1174 (1989)

[5] Kogler G, Radke TF, Lefort A, Sensken S, Fischer J, Sorg RV, Wernet P, Exp. Hematol., 33(5), 573 (2005)

[6] Ende M, Pac Med Surg, 74(2), 80 (1966)

[7] Kubanek B, Ferrari L, Tyler WS, Howard D, Jay S, Stohlman F, Blood, 32(4), 586 (1968)

[8] Stohlman F, Semin. Hematol., 4, 304 (1967)

[9] Metcalf D, Science, 254(5031), 529 (1991)

[10] Ihle JN, Chem. Immunol., 51, 65 (1992)

[11] Leary AG, Wong GG, Clark SC, Smith AG, Ogawa M, Blood, 75(10), 1960 (1990)

[12] Socinski MA, Cannistra SA, Elias A, Antman KH, Schnipper L, Griffin JD, Lancet, 1(8596), 1194 (1988)

[13] Migliaccio G, Migliaccio AR, Adamson JW, Blood, 72(1), 248 (1988)

[14] Tomonaga M, Golde DW, Gasson JC, Blood, 67(1), 31 (1986)

[15] Clark SC, Kamen R, Science, 236(4806), 1229 (1987)

[16] Ohara A, Suda T, Saito M, Miura Y, Okabe T, Takaku F, Exp. Hematol., 15(6), 695 (1987)

[17] Desauvage FJ, Hass PE, Spencer SD, Malloy BE, Gurney AL, Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang WJ, Oles KJ, Hultgren B, Solberg LA, Goeddel DV, Eaton DL, Nature, 369(6481), 533 (1994)

[18] Bartley TD, Bogenberger J, Hunt P, et al., Cell, 77(7), 1117 (1994)

[19] Hannum C, Culpepper J, Campbell D, et al., Nature, 368(6472), 643 (1994)

[20] Lyman SD, James L, Vanden Bos T, et al., Cell, 75(6), 1157 (1993)

[21] Rennick D, Jackson J, Yang G, Wideman J, Lee F, Hudak S, Blood, 73(7), 1828 (1989)

[22] Hodgkin PD, Bond MW, O'Garra A, Frank G, Lee F, Coffman RL, Zlotnik A, Howard M, J. Immunol., 141(1), 151 (1988)

[23] Caracciolo D, Clark SC, Rovera G, Blood, 73(3), 666 (1989)

[24] Brandt J, Briddell RA, Srour EF, Leemhuis TB, Hoffman R, Blood, 79(3), 634 (1992)

[25] Brandt JE, Bhalla K, Hoffman R, Blood, 83(6), 1507 (1994)

[26] Lebkowski JS, Schain LR, Okarma TB, Stem Cells, 13(6), 607 (1995)

[27] Daley JP, Daley BM, Wysocki MG, Caligiuri MA, Biddle WC, Ex Vivo Expansion of Human Hematopoietic Progenitor Cells in Serum-free STEMPRO TM-34 Medium, pp. 62-67. vol. 18. Life Technologies, Gaithersburg, U. S. A. (1996)

[28] Piacibello W, Sanavio F, Garetto L, Severino A, Dane A, Gammaitoni L, Aglietta M, Leukemia, 12(5), 718 (1998)

[29] Balducci E, Azzarello G, Valenti MT, et al., Stem Cells, 21(1), 33 (2003)

[30] Mobest D, Mertelsmann R, Henschler R, Biotechnol. Bioeng., 60(3), 341 (1998)

[31] Poloni A, Giarratana MC, Firat H, et al., Hematol Cell Ther, 39(2), 49 (1997)

[32] Gilmore GL, DePasquale DK, Lister J, Shadduck RK, Exp. Hematol., 28(11), 1297 (2000)

[33] Shadduck RK, Gilmore GL, Lister J, Stem Cells, 18(2), 154 (2000)