This work was funded by the Richard Thomas Leukaemia Fund, the Lady Tata Memorial Trust, the Northwick Park Hospital Leukaemia Research Trust Fund and the National Institute of Health Research (NIHR), UK.

1. Scaffold Manufacture and Bio-functionalization of Scaffolds
<ol>
 <li>To fabricate PU scaffolds (pore size 100-250 mm, porosity 90-95%) in the form of Petri dish disks, use the thermally induced phase separation13 process by preparing a polymer solution (5wt% in Dioxan) followed by freezing and subsequent solvent sublimation (Figure 1A).</li>
 <li>Cut the scaffold disk into cubes of 0.5 x 0.5 x 0.5 mm prior to coating with ECM proteins (Figure 1B).</li>
 <li>Pre-wet the ...

<ol>
	<li>Orkin, S., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoiesis:+an+evolving+paradigm+for+stem+cell+biology.">Hematopoiesis: an evolving paradigm for stem cell biology.</a> Cell. 132, 631-644 (2008).</li><li>Spradling, A., Drummond-Barbosa, D., Kai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+find+their+niche.">Stem cells find their niche.</a> Nature. 414, 98-104 (2001).</li><li>Panoskaltsis, N., Mantalaris, A., Wu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+mimicry+of+bone+marrow+tissue+ex+vivo.">Engineering a mimicry of bone marrow tissue ex vivo.</a> J Biosci. Bioeng. 100, 28-35 (2005).</li><li>Lo Celso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-animal+tracking+of+individual+haematopoietic+stem/progenitor+cells+in+their+niche.">Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.</a> Nature. 457, 92-96 (2009).</li><li>Mantalaris, A., Bourne, P., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+osteoclasts+in+a+three-dimensional+bone+marrow+culture+system.">Production of human osteoclasts in a three-dimensional bone marrow culture system.</a> Biochem. Eng. J. 20, 189-196 (2004).</li><li>Placzek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+bioprocessing:+fundamentals+and+principles.">Stem cell bioprocessing: fundamentals and principles.</a> J. R. Soc. Interface. 6, 209-232 (2009).</li><li>Dexter, T., Testa, N., Prescott, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology. 14, 387-405 (1976).</li><li>Piacibello, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+growth+factor+requirement+of+primitive+cord+blood+hematopoietic+stem+cell+for+self-renewal+and+amplification+vs+proliferation+and+differentiation.">Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation.</a> Leukemia. 12, 718-727 (1998).</li><li>Yoshida, T., Takagi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+processing+engineering+for+ex+vivo+expansion+of+hematopoietic+cells:+a+review.">Cell processing engineering for ex vivo expansion of hematopoietic cells: a review.</a> Biochemical Engineering Journal. 20, 99-106 (2004).</li><li>Lim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intelligent+bioprocessing+for+haemotopoietic+cell+cultures+using+monitoring+and+design+of+experiments.">Intelligent bioprocessing for haemotopoietic cell cultures using monitoring and design of experiments.</a> Biotechnol. Adv. 25, 353-368 (2007).</li><li>Mortera-Blanco, T., Mantalaris, A., Bismarck, A., Panoskaltsis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+three-dimensional+scaffold+for+ex+vivo+biomimicry+of+human+acute+myeloid+leukaemia.">The development of a three-dimensional scaffold for ex vivo biomimicry of human acute myeloid leukaemia.</a> Biomaterials. 31, 2243-2251 (2010).</li><li>Mortera-Blanco, T., Mantalaris, A., Bismarck, A., Aqel, N., Panoskaltsis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+cytokine-free+expansion+of+cord+blood+mononuclear+cells+in+three-dimensional+scaffolds.">Long-term cytokine-free expansion of cord blood mononuclear cells in three-dimensional scaffolds.</a> Biomaterials. 32, 9263-9270 (2011).</li><li>Safinia, L., Datan, N., Hohse, M., Mantalaris, A., Bismarck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+methodology+for+the+effective+surface+modification+of+porous+polymer+scaffolds.">Towards a methodology for the effective surface modification of porous polymer scaffolds.</a> Biomaterials. 26, 7537-7547 (2005).</li></ol>

We have nothing to disclose.

The ex vivo 3D culture system presented here enables us to establish a 3D biomimicry of hematopoiesis that recapitulates the original BM architecture and cellular phenotype independent of exogenous cytokines. The 3D model provides the structure and the microenvironment that enables normal and abnormal hematopoietic cells to proliferate in conditions similar to those encountered in vivo.
The selection of the polymeric scaffold material represented a critical step in the biomi...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue No</td>
		</tr>
		<tr>
			<td>Dioxan</td>
			<td>Invitrogen</td>
			<td>D20,186-3</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Invitrogen</td>
			<td>12440-053</td>
		</tr>
		<tr>
			<td>Ficoll-Paque</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
		</tr>
		<tr>
			<td>MTS</td>
			<td>Promega</td>
			<td>G3580</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Fluka Biochemika</td>
			<td>49624</td>
		</tr>
		<tr>
			<td>Wright-Giemsa</td>
			<td>Sigma-Aldrich</td>
			<td>WG32</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10108-165</td>
		</tr>
		<tr>
			<td>CD71</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-32272</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
		</tr>
		<tr>
			<td>CD45-FITC</td>
			<td>BD Pharmigen</td>
			<td>74895</td>
		</tr>
		<tr>
			<td>CD71-PE</td>
			<td>BD Pharmigen</td>
			<td>555537</td>
		</tr>
		<tr>
			<td>CD235a-PE-Cy5</td>
			<td>BD Pharmigen</td>
			<td>555570</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S-8032</td>
		</tr>
	</tbody>
</table>

ex vivo mimicry of normal and abnormal human hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Watch this Scientific Journal Video about Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis at JoVE.com

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

ex-vivo-mimicry-of-normal-and-abnormal-human-hematopoiesis

Research

JoVE Journal

Bioengineering

15.4K visualizações.  Imperial College London. Um sistema de cultura de 3D para a hematopoiese é descrito usando sangue do cordão umbilical humano e células de medula óssea leucémicas. O método baseia-se na utilização de um poliuretano poroso sintético andaime revestido com proteínas da matriz extracelular. Este andaime é adaptável para acomodar uma grande variedade de células.