1Department of Chemical Engineering and Chemical Technology, South Kensington campus, Imperial College London, 2Department of Hematology, Northwick Park & St. Mark's campus, Imperial College London
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Mortera-Blanco, T., Rende, M., Macedo, H., Farah, S., Bismarck, A., Mantalaris, A., et al. Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis. J. Vis. Exp. (62), e3654, doi:10.3791/3654 (2012).
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.
1. Scaffold Manufacture and Bio-functionalization of Scaffolds
2. Mononuclear Cell Isolation and Scaffold Seeding
3. In situ Cell Proliferation and Morphology: MTS, SEM and Cytospins
4. Flow Cytometric Analysis of the Cellular Population
5. Representative Results
An example of hematopoietic cellular growth kinetics without the addition of exogenous growth factors is shown in Figure 2. Due to the heterogeneous nature of hematopoiesis, two different cells: normal and abnormal hematopoietic cells are illustrated. In Figure 2A, cellular proliferation of human CBMNC is evident after 28 days in culture. Figure 2B shows the growth kinetics in the mimicry using human primary leukemic cells. Cellular proliferation is assessed using the MTS assay which measures cellular metabolic activity in relation to absorbance. In both cases, the cells proliferated and established in the model. Differences in growth kinetics are observed; normal hematopoietic cells establish the culture faster than the leukemic cells.
The morphology of the harvested cells even after 28 days of culture in the absence of exogenous growth factors was typical of normal hematopoietic cells (Figure 3A) and leukemic cells (Figure 3B). Central sections of the scaffolds were analyzed by SEM after the scaffolds were removed from the culture and showed the spreading of the seeded cells throughout the scaffold, establishing themselves in clusters and in "niche-like" structures (Figure 3A' &B'). Figure C shows the pore size and distribution in an unseeded scaffold used as a control. Multiphoton microscopy after 28 days was used to highlight the distribution of the cells within the 3D scaffold in situ and it showed the presence of erythroid islands in central sections of the scaffold (Figure 4) by the expression of the marker CD71 which is positive in erythroblasts. This proves the importance of mature and maturing cells during erythropoiesis. Finally, flow cytometry graphs of the cells prior seeding shows the difference in the phenotype of hematopoietic cells, where Figure 5A represents normal hematopoietic cells: human CBMNCs and Figure 5B illustrates abnormal hematopoietic cells: primary leukemic cells. Levels of CD235a + and CD45+ corresponding to erythrocytes and leukocytes respectively are higher in the normal sample than in the leukemic highlighting the hemoblastic nature of the leukemias.
Figure 1. Illustration of the processes involved in PU scaffold manufacture and bio-functionalization. A) PU is dissolved in Dioxan (5wt%) and by the thermally induced phase separation process and subsequent solvent sublimation the scaffold is produced, as described by Safinia et al 13. B) The scaffold disk is then cut into cubes of 0.5 x 0.5 x 0.5 mm and then coated by centrifugation with extra cellular matrix (ECM) proteins. C) MNC are extracted from human umbilical CB or from BM aspiration using density gradient centrifugation and seeded (2x106 cells/scaffold) in the PU scaffolds with a micropipette.
Figure 2. Cellular proliferation measured using the MTS assay: A) using human cord blood MNCs; B) using human primary leukemic cells. The columns show the growth of cells over time when seeded in the PU scaffolds without the addition of exogenous cytokines.
Figure 3: Cell morphology and distribution around the scaffolds using cytospins, scanning electron micrographs. (A-B) Representative Wright-Giemsa stained cytospins of A) cord blood mononuclear cells collected from PU scaffolds after 28 days of culture, and B) bone aspirated leukemic cells collected from PU scaffolds after 14 days of culture. Both experiments were carried out in cytokine free condition. (A'-B') Representative central sections of the PU scaffold SEM micrographs of A') seeded cord blood MNCs and B') leukemic cells after being cultured for 28 days, and C) control scaffold with no cells.
Figure 4. Multiphoton micrograph of a PU scaffold seeded with cord blood MNCs after 28 d in culture and stained with the erythroid marker CD71.
Figure 5. Flow cytometry 3D dot-plots stained for CD45, CD71 and CD235a surface expression markers. The isotype control obtained is also presented for comparison of the relative fluorescence intensity. Panel A shows human cord blood mononuclear cells positive for the above markers; Panel B shows human primary leukemic cells.
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 biomimicry design. Important advantages in biomaterials have led to an increasing interest in polymers that mimic the in vivo environment by providing the required architecture, structural properties and necessary biosignals. The polymers must meet minimum requirements essential for their application in biomedicine because they are always in direct contact with living cells, which are sensitive to their immediate environment. In general, an ideal scaffold should be biocompatible, highly porous, with enough mechanical strength to maintain a defined 3D structure, high surface area per volume ratio and reproducible fabrication. PU comprises all those properties together although the high porosity and mechanical strength can be adapted and changed depending on the quenching temperature during the TIPS process. As a result, this biomimicry can be adapted to other types of cells by increasing or decreasing the porosity and pore size as needed.
The scaffold in this experiment is coated with the ECM protein collagen type I; this protein, together with other ECM proteins were tested previously with our 3D model 11. This showed that collagen type I accelerated cell adhesion and was selected for that reason. The coating can be modified by using different ECM proteins depending on the type of cells that are being studied. The advantage to incorporate the ECM proteins to the synthetic scaffolds is that not only they provide the cell-binding sequence for cell adhesion, but also offer secondary interactions with other ECM proteins and interactions with growth factors that stabilize the binding of the cells thus enhancing cell adhesion, which results in better cell growth and maturation.
Numerous studies have been done on ex vivo hematopoiesis using both 2D and 3D systems; and all of them include the addition of abnormally high concentrations of exogenous cytokines. These studies have helped to elucidate the molecular determinants of hematopoiesis, but the cellular and microenvironmental elements integral to this process have been difficult to decipher based on the limitations of these same techniques.
The method described here, a biomimicry of the BM made of a highly porous polymeric scaffold coupled with ECM coating is optimal for sustaining and stabilizing the growth of normal and abnormal hematopoiesis without the need of any addition of cytokines. The mimicry supports multi-lineal hematopoiesis rendering the system suitable for use as a platform for drug discovery and scientific study into mechanisms of normal and abnormal hematopoiesis ex vivo.
We have nothing to disclose.
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.
|PBS||GIBCO, by Life Technologies||14190-094|
|Fetal bovine serum||GIBCO, by Life Technologies||10108-165|
|CD71||Santa Cruz Biotechnology, Inc.||sc-32272|
|Alexa Fluor 488||Invitrogen||A11001|