In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.
Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.
Tissue culture was developed to study cell behavior in a controlled environment to minimize the systemic variability when comparing various processes in intact organisms. This method was first established in the early 19001,2 and refers to a technique where tissue explants were cultured ex vivo in a glass dish. In the mid-1900s the system was adapted to grow dispersed cells rather than fragments of intact tissues, and the terms 'tissue culture’ and 'cell culture’ became synonymous3. In such conventional cell culture systems cells are grown on the surface of the tissue culture plastic overlaid with growth medium supplemented with various growth factors. Two types of cultures have emerged based on the adhesion capacity of various cells: adherent cell culture, where cells attach and spread on the tissue culture plastic and nonadherent cultures, where cells are propagated in suspension. Since the early days of cell culture, multiple immortal cell lines have been created, such as HeLa4, the first human cancer cell line. These cell lines have the capacity to proliferate indefinitely in cell culture, and most do not require any special treatment to maintain viability.
The reductionist approach of the original cell culture methods was designed to simplify the system as much as possible by including only the bare minimum components required to sustain cell viability and proliferation. However, simplified cell culture approaches fail to support ex vivo most primary human cell types with finite life-span. Therefore, new culture systems are being designed to approximate the tissue microenvironment as closely as possible, thus allowing cell explants to grow under physiological conditions. In contrast to the conventional/reductionist cell culture approaches, 3-dimensional (3-D) culture systems are now becoming a preferred method to efficiently culture various human cell lines and primary cells to subsequently study mechanisms involved in health and disease, in the context of a supportive microenvironment. Such 3-D systems are usually set up using reconstructed matrices and/or medium supplemented with growth factors, in order to recapitulate the tissue microenvironment to study cell types of interest. The first of these 3-dimensional (3-D) culture models was developed to study mammary gland development. To provide the cells with native conditions mammary epithelial cells were embedded in Matrigel, collagen IV and laminin-rich source of extracellular matrix (ECM), and overlaid with growth medium. Under such conditions, the mammary epithelial cells formed clusters resembling the mammary acini, and upon stimulation with lactogenic hormones, these acini secreted casein, and other milk proteins, into the hollow lumena of the acini-like structures. Casein secretion was not observed in standard cultures even after addition of prolactin5, further emphasizing the role of microenvironment in preserving the morphological and phenotypic characteristics of cells. Another demonstration of the loss of normal cellular function when cells are taken out of the context of their physiological microenvironment is a demonstration that without supportive microenvironment, keratinocytes fail to form stratified epidermis6. A number of other 3-D models have been created to allow ex vivo propagation of primary cells7,8 .
The crucial role of microenvironment in cell behavior was elegantly demonstrated in a study where mammary epithelial cells formed "inside-out" acini when cultured in collagen I matrix, compared to the correctly polarized acini that were formed in Matrigel. This loss of proper morphology was reverted when laminin-producing myoepithelial cells were added to the collagen I cultures9. Moreover, correct microenvironment is required for the accurate genotype manifestation. Grown in a dish, MCF7 breast cancer cells transfected with a cell-cell adhesion molecule CEACAM1 behave exactly the same as the untransfected CEACAM negative cells. However, when cultured in Matrigel, in contact with ECM, wildtype MCF7 form tumor-like structures while MCF7 cells transfected with CEACAM1 revert to a normal phenotype and form acini with hollow lumena, as has been established with nonmalignant mammary epithelium10. Similarly, blocking β1-integrin in breast cancer cells does not change their behavior under standard culture conditions, but the same experiment performed in 3-D cultures demonstrates that blocking β1-integrin reverts malignant cells to a normal phenotype11. Therefore, tissue microenvironment is not only required to maintain cell viability, but also to retain proper cell function.
In addition to providing a system where cell behavior can be studied under physiological conditions, 3-D cultures function as robust and reliable medium for preclinical testing of novel therapeutics8,12,13. Culturing cells in 3-D allows screening of investigational compounds under the conditions of environment-mediated drug-resistance14, where the contribution of cell-cell and cell-ECM adhesion could be assessed. Furthermore, off-target toxicity of new compounds can be ascertained by incorporating multiple cellular compartments of various tissues. Such screens can be performed more rapidly and are more cost-effective than the comparable studies in vivo8.
Here we present a setup of a 3-D model of reconstructed bone marrow (rBM) where normal and malignant bone marrow (BM) cells proliferate ex vivo in a system closely mimicking the microenvironment of the human BM. Previous attempts at growing primary human BM cells in 2-D cultures, liquid or adherent cocultures with various components of BM stroma, or 3-D, semi-solid agar cultures have met with limited success due to their inability to supply the cells with the components of tissue microenvironment15-18. In these systems primary BM cells had poor viability and failed to proliferate ex vivo. Another system where human BM progenitor cells are grown in spheroid cocultures with BM stromal cells is a 3-D system where hematopoietic stem cell viability and proliferation was sustained for at least 96 hr19. However, although highly beneficial to the understanding of the hematopoietic progenitor biology, this system does not faithfully recapitulate the microenvironment of the BM owing to the absence of ECM components, therefore, limiting its usefulness. The rBM model described here presents a comprehensive system where both cellular and extracellular compartments of the human BM are reconstructed in vitro. We show that rBM cultures can support ex vivo growth of normal human BM cells, as well as cells isolated from patients with various hematological disorders.
1. Advance Preparation
2. Preparation of Reagents
3. Embedded 3-D Culture of Nonmalignant and Malignant Human BM Cells
4. 'In-matrix' Imaging
5. Isolation of Cells from rBM
Since primary bone marrow cells fail to proliferate under standard tissue culture conditions, the rBM system was created to closely mimic the bone microenvironment, providing a system to culture and expand BM cells ex vivo. The major components of the BM extracellular matrix, fibronectin, collagen I, collagen IV, and laminin21, were incorporated into rBM matrix to provide the structural scaffold to this 3-D culture. Endosteum, the interface between the bone and the BM, rich in collagen I and fibronectin, was reconstructed by coating the surface of a tissue culture dish with these proteins. Crosstalk between multiple cellular compartments within the BM contributes to the overall homeostasis of the tissue. To ensure that none of the cellular components are left out, the rBM culture was set up using unfractionated mononuclear cells from the human BM needle aspirate biopsies. To ensure that the system is supplied with hormones, growth factors, and cytokines present in the circulatory environment, the culture medium was supplemented with human plasma corresponding to the specimen being cultured (i.e. plasma from normal donors was added when nonmalignant BM cells were placed in rBM, while plasma from patients was added to the cultures of malignant cells) (Figure 1).
BM and blood samples from healthy donors and patients with various hematological malignancies were collected after approval from Purdue University and University of Alberta Institutional Research Boards and after written informed consent in accordance with the Declaration of Helsinki (Table 1). Mononuclear cells isolated from BM biopsies proliferate and maintain their viability in rBM system for up to 30 days (Figure 2). The cell concentration of 0.5 x 106 cells/100μl of rBM matrix has been determined to be the optimum density for primary BM cells. Going beyond this density overcrowds the system decreasing cell viability and proliferation rates over time. A lower density has also been shown to be detrimental to the overall heath of the system as cell-cell contacts have proven vital for a robust cell growth. Growth within the rBM can be followed after labeling cells with carboxyfluorescein diacetate, succinimidyl ester (CFSE). Fluorescence intensity of CFSE halves with each cell division, thus cell proliferation can be tracked by microscopy or flow cytometry over time. Moreover, the cellular compartments within the rBM culture are maintained in the same proportions as were present in the biopsy samples8. In addition to the cells of the hematopoietic lineages, stromal compartments exhibit robust growth in rBM. Alkaline phosphatase expressing osteoblasts capable of mineralizing calcium, tartrate resistant acid phosphatase staining osteoclasts, oil red positive adipocytes, and fibronectin producing stromal cells are found at the endosteum/BM interface of the rBM system (Figure 3).
Taken together, our data demonstrate that the rBM model provides a first ex vivo system where primary human BM cells from healthy donors and patients with various hematological malignancies such as leukemia, lymphoma, and multiple myeloma can be sustained and expanded for up to 30 days. rBM provides a comprehensive model to study cell behavior under physiological conditions in the context of the native BM microenvironment. This system has been used by our laboratory to identify the phenotype of the multiple myeloma cancer stem cells and to study the interactions between the malignant cells and the BM extracellular matrix as well as the cross-talk between the malignant cells and the BM stroma.
Diagnosis | Cell type | Survival | Proliferation |
Nonmalignant | BM | Moderate | Moderate |
Amyloidosis | BM | High | High |
Monoclonal gammopathy of undetermined significance | BM | High | High |
PBMC | No | No | |
Plasmacytoma | BM | High | High |
Smoldering multiple myeloma | BM | High | High |
Multiple myeloma | BM | High | High |
PBMC | No | No | |
MB | High | High | |
Plasma cell leukemia | BM | High | High |
Waldenstroms macroglobulenemia | BM | High | High |
Acute myelogenous leukemia | BM | High | High |
Acute promyelocytic leukemia | BM | High | High |
PBMC | No | No | |
Acute lymphocytic leukemia | BM | Moderate | Moderate |
Chronic myelogenous leukemia | BM | Moderate | High |
Chronic lymphocytic leukemia | BM | Low | Slow |
PBMC | No | No | |
Non-Hodgkin’s lymphoma | BM | Moderate | Slow |
PBMC | No | No |
Table 1. Survival and proliferation of various specimens cultured in rBM. Bone marrow mononuclear cells (BM), peripheral blood mononuclear cells (PBMC), or mobilized blood samples (MB) from healthy donors and patients diagnosed with various malignancies were cultured in rBM. The table lists all specimen types that were cultured in rBM along with the cell type (i.e. BM, PBMC, or MB). The extent of cell survival and proliferation in rBM are noted. While BM and MB exhibit robust growth in rBM, PBMCs isolated from patients with various malignancies were not viable in rBM.
Figure 1. Schematic representation of rBM setup. rBM consists of two phases: 1) the endosteal coating (fibronectin, collagen I (CI)) and 2) rBM matrix (fibronectin, collagen I (CI), collagen IV (CIV) and laminin). Note: collagen IV and laminin are the major components of Matrigel, thus no separate addition of these proteins is required. These phases are set up in two steps by overlaying the rBM matrix/cell mixture on top of the thin coating of endosteal matrix. To follow cell proliferation over time, cells can be labeled with CFSE prior to mixing with the rBM matrix. Over the duration of the culture, cells can be visualized by microscopy and cell migration can be followed in real time using a microscope equipped with a temperature/CO2 controlled chamber. After 14-30 days in culture cells can be isolated from the matrix and subjected to a variety of downstream analyses, including both in vitro and in vivo procedures. Click here to view larger image.
Figure 2. Time course of self-segregation of cellular compartments in rBM. BM cells were grown in rBM for the indicated number of days. Stromal elements become evident by day 14, but can be detected as early as day 5 in rBM. Over the course of the culture period cells can be seen migrating through rBM matrix and aggregating into distinct clusters. Masses of malignant cells expand over time (compare cluster sizes between day 9-30). Representative view of the culture at each time point was captured using bright field microscopy on an inverted microscope (magnification: 200X). Click here to view larger image.
Figure 3. Stromal compartments are ‘supplied’ within the rBM. To evaluate the composition of the stromal components that outgrow in rBM, cells at the transition between rBM matrix and endosteal coating were evaluated for fibroblast, osteoblast, adipocyte, and osteoclast-specific morphology and marker expression. (a) To detect fibroblasts, cells were stained for fibronectin (green), actin (red), and nuclei (blue) and imaged on a confocal microscope (magnification: 200X). (b) Cells with stromal morphology were shown to be preosteoblasts with high levels of alkaline phosphatase activity (b.1) and capable of mineralizing calcium as determined by Alizarin Red staining (b.2). (c) Cells with a visible presence of lipid droplets were identified as adipocytes based on positive staining with Oil Red (c.1). (d) Cells with occasional multiple nuclei were recognized as preosteoclasts and were positive for tartarate resistant acid phosphatase (TRAP) (d.1). Bright field and color (nonfluorescent) images were acquired on an inverted microscope equipped with a color camera (magnification: 200X). Click here to view larger image.
The rBM model provides a comprehensive system where cellular composition of the BM is maintained ex vivo for up to 30 days. BM cells grown in rBM self-stratify according to their function closely mimicking the BM architecture found in vivo. Moreover, this is the first system that allows for the expansion of the multiple myeloma clone8. The most critical steps for ensuring the robust growth of BM cells and the overall success of the culture are: 1) to obtain a healthy population of the BM cells with >70% viability and mostly free of red blood cells, 2) to ensure that the matrix components are mixed well and that the cells are distributed uniformly throughout the matrix, and 3) to take care not to damage the matrix when growth medium is added to system. To ensure the robust proliferation of malignant cells, growth medium should be supplemented with human plasma matching the diagnosis of the patient whose cells are being cultured. One limitation of the rBM system that was noticed is its inability to support purified populations of CD34+ hematopoietic stem cells, CD20+ B cells, and CD138+ plasma cells without the stromal compartment of the BM.
The rBM system is highly versatile and can be modified in many ways to best suit the goals of specific studies. Endothelial compartment can be added to the system to mimic the vascularization of the BM and additional extracellular matrix components can be supplied to bring the matrix composition as close as possible to the in vivo make-up of the BM and the matrix concentrations can be adjusted to reflect the disease states where the concentrations of individual matrix components may be altered21. In addition to culturing primary cells, rBM system is suitable for coculturing various cell lines to study cell-cell interactions between specific cell populations. For example, the system can be set up as a coculture where stromal cells are plated over the endosteal coating, hematopoietic cells are added on top of the stromal cells, and the entire stromal/hematopoietic coculture is overlaid first with the rBM matrix and then with the growth medium. Also useful, could be the modifications of the medium composition to include plasma from various sources or medium conditioned by stroma to study how soluble factors affect cell behavior12. Cytokines, growth factors, and hormones can also be added to the growth medium, however, care should be taken not to replace all growth medium in the culture at once as growth sustaining factors that were secreted by the cells in rBM may be lost, thus, affecting the overall viability of the system. We suggest replacing up to 50% of the medium at a time.
The rBM system can be used to study both cell behavior (i.e. proliferation, viability, migration, etc.) and to assess the potency and off-target effects of novel therapeutics8,12 . The system is set up to recapitulate the tissue microenvironment, thus, the efficacy of investigational drugs can be evaluated under the conditions of environment-mediated drug-resistance14. Using live-cell real-time microscopy, cell viability over the course of treatment can be assessed by live/dead staining and the affect of treatment on various organelles can be evaluated using multiple commercially available organelle-specific dyes that readily penetrate the rBM matrix and are taken-up by the cells without generating significant background that could affect imaging.
The rBM system provides a highly versatile and adaptable model to study BM disorders. The benefit of such 3-D culture method compared to the standard 2-D cultures is in the ability to study cell behavior in the context of the tissue microenvironment ensuring that the effects of the multiple interactions are not missed when cells are isolated from their physiological environment and placed in a plastic dish. The appeal of the rBM system over in vivo models lies in the transparency of the system where the interactions that cannot be seen in vivo due to the limitations of imaging techniques could be easily dissected. The rBM system provides a medium for easy manipulation of components while maintaining the overall tissue composition, thus allowing the researcher to dissect the molecular mechanisms under investigation. Moreover, the rBM system has been validated as a cost-effective way, compared to in vivo testing, to conduct preclinical studies of novel therapeutics8,12. Taken together, the rBM model provides a system where many aspects of tissue biology can be investigated ex vivo using primary specimens.
The authors have nothing to disclose.
This work was funded by grants from the National Institutes of Health, National Cancer Institute (1R21CA141039), BD Biosciences Research Grant Award, the American Cancer Society Institutional Research Grant (IRG #58-006-53), to the Purdue University Center for Cancer Research, and the Canadian Institutes of Health Research and the Alberta Cancer Board Research Initiatives Program. M.P. was supported by the National Institutes of Health, National Cancer Institute, Cancer Prevention Internship Program (R25CA128770; PI: D. Teegarden) administered by the Oncological Sciences Center and the Discovery Learning Research Center at Purdue University.
CellTrace CFSE proliferation kit-for flow cytometry | Invitrogen/Life technologies | C34554 | |
Fibronectin from human plasma | Millipore | NG1884448 | |
Ficoll-Paque PLUS | GE Lifesciences | 17-1440-02 | |
Matrigel basement membrane matrix | BD Biosciences | 354234 | |
Rat tail collagen type I | BD Biosciences | 354249 | Collagen I from Millipore does not polimerize properly using this protocol |
Vectashield mounting medium for fluorescence (regular set) | Vector Laboratories | H-1000 | |
SigmaFast Red TR/Napthol AS-MX tablets | Sigma-Aldrich | F4648 | For TRAP staining |
SigmaFast BCIP/NBT tablets | Sigma-Aldrich | B5655 | For alkaline phosphatase staining |
Zeiss confocal LSM 510 microscope | Carl Zeiss | ||
Zeiss 510 software | Carl Zeiss | Image analysis software for confocal analysis | |
Zeiss Axiovert 200M inverted microscope | Carl Zeiss | ||
MetaMorph software | Molecular Devices | Image analysis software for Axiovert 200M | |
FACSort flow cytometer | BD biosciences | ||
CellQuest Pro software | BD biosciences | Software for the analysis of flow cytometry data |