A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Summary

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.

Abstract

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.

Introduction

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 1900^1,2 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 supple....

Protocol

1. Advance Preparation

1. Keep sterile serological pipettes and pipette tips at 4 °C.
   Troubleshooting: Matrigel gels at room temperature (RT); using cold pipettes and tips will prevent Matrigel from gelling during the culture setup.
2. Thaw a bottle of Matrigel at 4 °C overnight.

2. Preparation of Reagents

1. Preparation of fibronectin stock solution: Make a 1 mg/ml stock solution of fibronectin by dissolving 100 mg human fibronectin in 100 ml of distilled water. Once the fibronectin has dissolved, sterile filter, aliquot, and store at 4 °C for up to 6 months.
   

Results

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 fibronect.......

Discussion

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 cell.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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