Skip to main content
SpringerLink
Log in

Stromalized microreactor supports murine hematopoietic progenitor enrichment

  • Published:
Biomedical Microdevices Aims and scope Submit manuscript

Abstract

There is an emerging need to process, expand, and even genetically engineer hematopoietic stem and progenitor cells (HSPCs) prior to administration for blood reconstitution therapy. A closed-system and automated solution for ex vivo HSC processing can improve adoption and standardize processing techniques. Here, we report a recirculating flow bioreactor where HSCs are stabilized and enriched for short-term processing by indirect fibroblast feeder coculture. Mouse 3 T3 fibroblasts were seeded on the extraluminal membrane surface of a hollow fiber micro-bioreactor and were found to support HSPC cell number compared to unsupported BMCs. CFSE analysis indicates that 3 T3-support was essential for the enhanced intrinsic cell cycling of HSPCs. This enhanced support was specific to the HSPC population with little to no effect seen with the Lineagepositive and Lineagenegative cells. Together, these data suggest that stromal-seeded hollow fiber micro-reactors represent a platform to screening various conditions that support the expansion and bioprocessing of HSPCs ex vivo.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • M. Bhatia, D. Bonnet, U. Kapp, J.C.Y. Wang, B. Murdoch, J.E. Dick, Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J. Exp. Med. 186, 619–624 (1997)

    Article  Google Scholar 

  • E. Conneally, J. Cashman, A. Petzer, C. Eaves, Expansion In Vitro of Transplantable Human Cord Blood Stem Cells Demonstrated Using a Quantitative Assay of their Lympho-Myeloid Repopulating Activity in Nonobese Diabetic–Scid/Scid Mice. Proc. of the Natl. Acad. of Sci. 94, 9836–9841 (1997)

    Article  Google Scholar 

  • T.R. Coughlin, G.L. Niebur, Fluid shear stress in trabecular bone marrow due to low-magnitude high-frequency vibration. J. Biomech. 45, 2222–2229 (2012)

    Article  Google Scholar 

  • T.A. Davis, W. Wiesmann, W. Kidwell, T. Cannon, L. Kerns, C. Serke, T. Delaplaine, A. Pranger, K.P. Lee, Effect of spaceflight on human stem cell hematopoiesis: Suppression of erythropoiesis and myelopoiesis. J. Leukoc. Biol. 60, 69–76 (1996)

    Article  Google Scholar 

  • A. De Leon, H. Mayani, O.T. Ramirez, Design, characterization and application of a minibioreactor for the culture of human hematopoietic cells under controlled conditions. Cytotechnology 28, 127–138 (1998)

    Article  Google Scholar 

  • C. Delaney, B. Varnum-Finney, K. Aoyama, C. Brashem-Stein, I.D. Bernstein, Dose-dependent effects of the notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells. Blood 106, 2693–2699 (2005)

    Article  Google Scholar 

  • C. Delaney, S. Heimfeld, C. Brashem-Stein, H. Voorhies, R.L. Manger, I.D. Bernstein, Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution. Nat. Med. 16, 232–236 (2010)

    Article  Google Scholar 

  • M.J. Domingues, S.K. Nilsson, B. Cao, New agents in HSC mobilization. Int. J. Hematol. 105, 141–152 (2017)

    Article  Google Scholar 

  • S.P. Dormady, X.-M. Zhang, R.S. Basch, Hematopoietic progenitor cells grow on 3T3 fibroblast monolayers that overexpress growth arrest-specific gene-6 (GAS6). Proc. Natl. Acad. Sci. 97, 12260–12265 (2000)

    Article  Google Scholar 

  • K. Futrega, K. Atkinson, W.B. Lott, M.R. Doran, Spheroid Coculture of hematopoietic stem/progenitor cells and monolayer expanded mesenchymal stem/stromal cells in polydimethylsiloxane microwells modestly improves in vitro hematopoietic stem/progenitor cell expansion. Tissue Engineering Part C, Methods 23, 200–218 (2017)

    Article  Google Scholar 

  • J.L. Gori, J.M. Butler, B. Kunar, M.G. Poulos, M. Ginsberg, D.J. Nolan, Z.K. Norgaard, J.E. Adair, S. Rafii, H.-P. Kiem, Endothelial cells promote expansion of long-term engrafting marrow hematopoietic stem and progenitor cells in primates. Stem Cells Transl. Med. 6, 864–876 (2017)

    Article  Google Scholar 

  • H.A. Himburg, G.G. Muramoto, P. Daher, S.K. Meadows, J.L. Russell, P. Doan, J.T. Chi, A.B. Salter, W.E. Lento, T. Reya, et al., Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells. Nat. Med. 16, 475–482 (2010)

    Article  Google Scholar 

  • A.A. Irani, S.S. Craig, G. Nilsson, T. Ishizaka, L.B. Schwartz, Characterization of human mast cells developed in vitro from fetal liver cells cocultured with murine 3T3 fibroblasts. Immunology 77, 136–143 (1992)

    Google Scholar 

  • J. Kiernan, P. Damien, M. Monaghan, R. Shorr, L. McIntyre, D. Fergusson, A. Tinmouth, D. Allan, Clinical studies of ex vivo expansion to accelerate engraftment after umbilical cord blood transplantation: A systematic review. Transfus. Med. Rev. 31, 173–182 (2017)

    Article  Google Scholar 

  • M.R. Koller, J.G. Bender, W.M. Miller, E.T. Papoutsakis, Expansion of primitive human hematopoietic progenitors in a perfusion bioreactor system with IL-3, IL-6, and stem cell factor. Biotechnology (NY) 11, 358–363 (1993a)

    Article  Google Scholar 

  • M.R. Koller, S.G. Emerson, B.O. Palsson, Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures. Blood 82, 378–384 (1993b)

    Google Scholar 

  • S. Kumar, H. Geiger, HSC niche biology and HSC expansion ex vivo. Trends Mol. Med. 23, 799–819 (2017)

    Article  Google Scholar 

  • M.G. Levee, G.M. Lee, S.H. Paek, B.O. Palsson, Microencapsulated human bone marrow cultures: A potential culture system for the clonal outgrowth of hematopoietic progenitor cells. Biotechnol. Bioeng. 43, 734–739 (1994)

    Article  Google Scholar 

  • F. Levi-Schaffer, K.F. Austen, J.P. Caulfield, A. Hein, P.M. Gravallese, R.L. Stevens, Co-culture of human lung-derived mast cells with mouse 3T3 fibroblasts: Morphology and IgE-mediated release of histamine, prostaglandin D2, and leukotrienes. J. Immunol.. (Baltimore, md: 1950) 139, 494–500 (1987)

    Google Scholar 

  • W.C. Liles, H.E. Broxmeyer, E. Rodger, B. Wood, K. Hübel, S. Cooper, G. Hangoc, G.J. Bridger, G.W. Henson, G. Calandra, et al., Mobilization of hematopoietic progenitor cells in healthy volunteers by AMD3100, a CXCR4 antagonist. Blood 102, 2728 (2003)

    Article  Google Scholar 

  • N. Liu, R. Zang, S.-T. Yang, Y. Li, Stem cell engineering in bioreactors for large-scale bioprocessing. Engineering in Life Sciences 14, 4–15 (2014)

    Article  Google Scholar 

  • E. Lorenz, D. Uphoff, T.R. Reid, E. Shelton, Modification of irradiation injury in mice and guinea pigs by bone marrow injections. J. Natl. Cancer Inst. 12, 197–201 (1951)

    Google Scholar 

  • W.C. Lui, Y.F. Chan, L.C. Chan, R.K. Ng, Cytokine combinations on the potential for ex vivo expansion of murine hematopoietic stem cells. Cytokine 68, 127–132 (2014)

    Article  Google Scholar 

  • P. Meissner, B. Schroder, C. Herfurth, M. Biselli, Development of a fixed bed bioreactor for the expansion of human hematopoietic progenitor cells. Cytotechnology 30, 227–234 (1999)

    Article  Google Scholar 

  • S. Méndez-Ferrer, T.V. Michurina, F. Ferraro, A.R. Mazloom, B.D. MacArthur, S.A. Lira, D.T. Scadden, A. Ma’ayan, G.N. Enikolopov, P.S. Frenette, Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466, 829–834 (2010)

    Article  Google Scholar 

  • S.J. Morrison, D.T. Scadden, The bone marrow niche for haematopoietic stem cells. Nature 505, 327–334 (2014)

    Article  Google Scholar 

  • S. Obi, K. Yamamoto, J. Ando, Effects of shear stress on endothelial progenitor cells. J. Biomed. Nanotechnol. 10, 2586–2597 (2014)

    Article  Google Scholar 

  • B.O. Palsson, S.H. Paek, R.M. Schwartz, M. Palsson, G.M. Lee, S. Silver, S.G. Emerson, Expansion of human bone marrow progenitor cells in a high cell density continuous perfusion system. Biotechnology (NY) 11, 368–372 (1993)

    Article  Google Scholar 

  • X. Pan, Q. Sun, Y. Zhang, H. Cai, Y. Gao, Y. Shen, W. Zhang, Biomimetic macroporous PCL scaffolds for ex vivo expansion of cord blood-derived CD34+ cells with feeder cells support. Macromol. Biosci. 17, 1700054 (2017)

    Article  Google Scholar 

  • S.R. Panch, J. Szymanski, B.N. Savani, D.F. Stroncek, Sources of hematopoietic stem and progenitor cells and methods to optimize yields for clinical cell therapy. Biol. of Blood and Marrow Transplant. 23, 1241–1249 (2017)

    Article  Google Scholar 

  • P. Peris, M.M. Roforth, K.M. Nicks, D. Fraser, K. Fujita, R.L. Jilka, S. Khosla, U. McGregor, Ability of circulating human hematopoietic lineage negative cells to support hematopoiesis. J. Cell. Biochem. 116, 58–66 (2015)

    Article  Google Scholar 

  • S. Perucca, A. Di Palma, P.P. Piccaluga, C. Gemelli, E. Zoratti, G. Bassi, E. Giacopuzzi, A. Lojacono, G. Borsani, E. Tagliafico, et al., Mesenchymal stromal cells (MSCs) induce ex vivo proliferation and erythroid commitment of cord blood haematopoietic stem cells (CB-CD34+ cells). PLoS One 12, e0172430 (2017)

    Article  Google Scholar 

  • J. Rak, K. Foster, K. Potrzebowska, M.S. Talkhoncheh, N. Miharada, K. Komorowska, T. Torngren, A. Kvist, Å. Borg, L. Svensson, et al., Cytohesin 1 regulates homing and engraftment of human hematopoietic stem and progenitor cells. Blood 129, 950–958 (2017)

    Article  Google Scholar 

  • R.A. Roberts, E. Spooncer, E.K. Parkinson, B.I. Lord, T.D. Allen, T.M. Dexter, Metabolically inactive 3T3 cells can substitute for marrow stromal cells to promote the proliferation and development of multipotent haemopoietic stem cells. J. Cell. Physiol. 132, 203–214 (1987)

    Article  Google Scholar 

  • C.E. Sandstrom, J.G. Bender, W.M. Miller, E.T. Papoutsakis, Development of novel perfusion chamber to retain nonadherent cells and its use for comparison of human "mobilized" peripheral blood mononuclear cell cultures with and without irradiated bone marrow stroma. Biotechnol. Bioeng. 50, 493–504 (1996)

    Article  Google Scholar 

  • C.A. Sardonini, Y.-J. Wu, Expansion and differentiation of human hematopoietic cells from static cultures through small-scale bioreactors. Biotechnol. Prog. 9, 131–137 (1993)

    Article  Google Scholar 

  • E. Schmelzer, A. Finoli, I. Nettleship, J.C. Gerlach, Long-term three-dimensional perfusion culture of human adult bone marrow mononuclear cells in bioreactors. Biotechnol. Bioeng. 112, 801–810 (2015)

    Article  Google Scholar 

  • E.J. Shpall, R. Quinones, R. Giller, C. Zeng, A.E. Baron, R.B. Jones, S.I. Bearman, Y. Nieto, B. Freed, N. Madinger, et al., Transplantation of ex vivo expanded cord blood. Biol. of Blood and Marrow Transplant. 8, 368–376 (2002)

    Article  Google Scholar 

  • J.E. Wagner, C.G. Brunstein, A.E. Boitano, T.E. DeFor, D. McKenna, D. Sumstad, B.R. Blazar, J. Tolar, C. Le, J. Jones, et al., Phase I/II trial of StemRegenin-1 expanded umbilical cord blood hematopoietic stem cells supports testing as a stand-alone graft. Cell Stem Cell 18, 144–155 (2016)

    Article  Google Scholar 

  • T.Y. Wang, J.K. Brennan, J.H. Wu, Multilineal hematopoiesis in a three-dimensional murine long-term bone marrow culture. Exp. Hematol. 23, 26–32 (1995)

    Google Scholar 

  • C. Xue, K.Y.C. Kwek, J.K.Y. Chan, Q. Chen, M. Lim, The hollow fiber bioreactor as a stroma-supported, serum-free ex vivo expansion platform for human umbilical cord blood cells. Biotechnol. J. 9, 980–989 (2014)

    Article  Google Scholar 

  • P.W. Zandstra, C.J. Eaves, J.M. Piret, Expansion of hematopoietic progenitor cell populations in stirred suspension bioreactors of normal human bone marrow cells. Biotechnology (NY) 12, 909–914 (1994)

    Google Scholar 

  • C.C. Zhang, M. Kaba, G. Ge, K. Xie, W. Tong, C. Hug, H.F. Lodish, Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells. Nat. Med. 12, 240–245 (2006)

    Article  Google Scholar 

  • E. Zonari, G. Desantis, C. Petrillo, F.E. Boccalatte, M.R. Lidonnici, A. Kajaste-Rudnitski, A. Aiuti, G. Ferrari, L. Naldini, B. Gentner, Efficient ex vivo engineering and expansion of highly purified human hematopoietic stem and progenitor cell populations for gene therapy. Stem Cell Reports 8, 977–990 (2017)

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported in part by the Shriners Hospitals for Children (B.P.), National Institutes of Health Grants R01EB012521 (B.P.). BP is a founder and equity holder of Sentien Biotechnologies, Inc. whom have licensed patents pertaining to MSC and extracorporeal device-based therapeutics. The authors declare no other competing financial interests.

Author information

Authors and Affiliations

  1. Department of Surgery, Center for Surgery, Innovation, & Bioengineering, Massachusetts General Hospital, Harvard Medical School and the Shriners Hospitals for Children, Boston, MA, 02114, USA

    Danika Khong, Matthew Li, Amy Singleton, Ling-Yee Chin & Biju Parekkadan

  2. Department of Biomedical Engineering, Rutgers University and the Department of Medicine, Rutgers Biomedical and Health Sciences, Piscataway, NJ, 08854, USA

    Biju Parekkadan

  3. Harvard Stem Cell Institute, Cambridge, MA, 02138, USA

    Biju Parekkadan

Authors
  1. Danika Khong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Matthew Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Amy Singleton
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ling-Yee Chin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Biju Parekkadan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Biju Parekkadan.

Electronic supplementary material

Fig. S1

LSK proportion of scatter in 2-D transwell co-cultures for both high and low stromal doses. All values are means + standard deviation. Data is representative of three biological replicates. * indicates p-values compared to BM alone. *p < 0.05, **p < 0.01, and ***p ≤ 0.0001. (GIF 31 kb)

High resolution image (EPS 730 kb)

Fig. S2

Various flow rates on cell number and viability. All values are means + standard deviation. Data is representative of three biological replicates. * indicates p-values compared to BM alone. *p < 0.05, **p < 0.01, and ***p ≤ 0.0001. (GIF 26 kb)

High resolution image (EPS 711 kb)

Fig. S3

Raw counts and viabilities for both high and low cell-seeded models. All values are means + standard deviation. Data is representative of three biological replicates. * indicates p-values compared to BM alone. *p < 0.05, **p < 0.01, and ***p ≤ 0.0001. (GIF 47 kb)

High resolution image (EPS 917 kb)

Fig. S4

Raw counts and viabilities for both high and low stromal dose for 3 T3-seeded micro-reactors. All values are means + standard deviation. Data is representative of three biological replicates. * indicates p-values compared to BM alone. *p < 0.05, **p < 0.01, and ***p ≤ 0.0001. (GIF 45 kb)

High resolution image (EPS 878 kb)

Fig. S5

LSK proportion and cell numbers for low stromal dose model. All values are means + standard deviation. Data is representative of three biological replicates. * indicates p-values compared to BM alone. *p < 0.05, **p < 0.01, and ***p ≤ 0.0001. (GIF 49 kb)

High resolution image (EPS 822 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Khong, D., Li, M., Singleton, A. et al. Stromalized microreactor supports murine hematopoietic progenitor enrichment. Biomed Microdevices 20, 13 (2018). https://doi.org/10.1007/s10544-017-0255-3

Download citation

  • Published:

  • DOI: https://doi.org/10.1007/s10544-017-0255-3

Keywords

Search

Navigation