Although three dimensional (3-D) cell culture systems have numerous advantages over traditional monolayer culture, the currently available 3-D cell culture media are cost-prohibitive for regular use by the majority of research laboratories. Here we show a simple system based on avian egg white that supports growth of cells in 3-D, at a significantly decreased cost. Specifically, we show that growth of immortalized human breast epithelial cells (MCF10A) in egg white–based medium results in formation of acini with hollow lumens, apoptotic clearance of the cells in the lumen, and apicobasal polarization comparable to what has been described using established 3-D culture media such as reconstituted basement membrane preparations (BM). There was no significant difference in MCF10A proliferation and acinar size between egg white and BM. We also cultured different established cell lines, oncogene-transformed MCF10A, and mouse mammary epithelial cells in egg white and BM, and observed similar morphology. In summary, our data convincingly argue that egg white can be used as a suitable alternative model for 3-D cell culture studies. We strongly believe that this simple and inexpensive method should allow researchers to perform 3-D cell culture experiments on a regular basis, and result in a dramatic increase of use of the 3-D cell culture in research. Thus, this finding lays the foundation for significantly increased, cost-effective use of 3-D cultures in cell biology.

Introduction

Over the last few years, much attention has been paid to the development and use of three dimensional (3-D) cell culture systems in order to replace the classical two-dimensional (2-D) monolayer system. Cells grown on flat 2-D tissue culture substrates can differ considerably in their morphology, differentiation, and cell-cell and cell-matrix interactions from those growing in more physiological 3-D environments (1). There are a growing number of studies that report differences in phenotype, cellular signaling, cell migration, and drug responses when the same cells are grown under 2-D or 3-D culture conditions (2). 3-D cultures are currently used in a broad range of cell biology studies, including tumor biology, cell adhesion, cell migration, and epithelial morphogenesis (3). Commonly used 3-D culture media contain a reconstituted basement membrane preparation derived from Engelbreth-Holm-Swarm (EHS) sarcoma, a mouse tumor rich in extracellular matrix (ECM) proteins. Unfortunately, the cost of all currently available 3-D media is extremely high, which prohibits their frequent use in the majority of academic research settings.

In our search for a reliable and economically viable replacement for the expensive commercial 3-D media, we hypothesized that avian egg white could be a potential alternative based on the following rationales: (i) It is sufficient to support growth and development of normal cells into a baby chick without any additional external nutrient support; (ii) the viscosity of egg white can be changed with temperature; (iii) egg white is transparent, which allows easy monitoring of growing cells; and (iv) it is inexpensive and easily available.

To demonstrate that egg white can indeed function as ECM and support growth of cells in 3-D, we used the nontransformed immortalized human breast epithelial cell line MCF10A as an example, since its growth in 3-D has been very well characterized. In 3-D, MCF10A cells recapitulate numerous features of normal breast epithelium in vivo, including the formation of spheroids resembling acini with a hollow lumen, and apical-basal polarization (4). This is in stark contrast to fully transformed epithelial cells, which do not develop tightly organized acinar structures but instead form large structures that lack organized adherent junctions and a central lumen and that fail to undergo growth arrest (5).

Materials and Methods

Preparation of Egg White–based Chamber Slide

We purchased chicken eggs from grocery stores or directly from the farm. After cleaning the outer shell of the egg using 70% alcohol, we made a small hole in the shell using sterilized metal forceps or a sterile pipet under a laminar flow hood. We then poured the egg white into a 50 ml falcon tube to ensure complete separation from egg yolk. To prevent the egg yolk from mixing with egg white, it is important to avoid removing the egg white completely from the shell. From one chicken egg, approximately 25 to 30 ml of egg white can be obtained. Egg white can be aliquoted into 1.5 ml sterile microcentrifuge tubes and stored at −80°C until use.

For cell culture, we thawed the egg white to room temperature and pipeted 100 µl using a 1000 µl pipet tip into each well of an 8-well chamber slide. Because the egg white is very viscous and it may be difficult to pipet with a normal sharp pipet tip, cutting the tip may be helpful. The egg white should be added directly to the middle of the wells, using caution that the egg white does not touch the top of the side wall of the wells. Spilling of egg white on the top of the side walls may cause capillary rise of the medium from the well to the cover of the chamber through the side walls, which may result in drying of the wells and contamination.

We then heated the 8-well chamber containing the egg white at 60°C on a heating block of the type used for 1.5 ml microcentrifuge tubes for 30–60 min. We found that there should be a minimum of direct contact between the wells and the metal part of the heating block to avoid rapid heating of some areas of the chamber; thus, the slide should be positioned in such a way that the chamber wells mostly align with the wells of the heating block and are not directly on the metal part. Increased temperature may lead to denaturing of the protein, causing the egg white to become white in color. If this occurs, the affected chambers should not be used in subsequent experiments. Evaporated water from the egg white usually condenses on the chamber cover, so at regular intervals we would shake off the condensed water from the chamber cover under a sterile hood. After approximately 30–60 min, the egg white becomes a semi-solid that adheres to the bottom of the well. The time varies depending on the heating block and the thickness of the egg white. When most of the egg white had adhered to the chamber slide, we took the slide back to the sterile hood and brought it back to room temperature. It is important to remove the chamber from the heating block before the entire egg white becomes semi-solid because this may cause drying of the egg white in some areas and formation of a round dry area on which cells are not able to grow in the middle of the chamber. We washed out any unstuck egg white from the chamber by adding 500 µl of growth medium to the chamber and removing it slowly using a Pasteur pipet. This step is important because the cells could grow on the unstuck egg white and will be lost later while changing the medium. We would either use the chamber right away for cell culture, or store it at 4°C overnight after adding 0.5 ml of sterile PBS or growth medium to each well.