Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Summary

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Abstract

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States¹. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae^2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.

Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation⁴.

Alginate hydrogels can be used to support the growth of many types of tissues⁵. Alginate is a linear polysaccharide composed of repeating units of β-D-mannuronic acid and α-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues^6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling⁵. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.

A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Protocol

1. Ovarian dissection and tissue isolation

1. Prepare a 0.5% (w/v) solution of sodium alginate, prepare from a modified version of the protocol described previously⁵, using sterile PBS and heat to 37°C. Mix by inversion or rocking, but do not vortex. Draw the alginate into a 1 ml syringe with a 25 G needle and maintain at 37°C.
2. Prepare 10mM sterile CaCl₂ solution for crosslinking the alginate upon tissue encapsulation and warm to 37°C.
3. Prepare 1mL of culture medium (αMEM + 5 units penicillin and 5 μg/ml streptomycin) per well in a 24 well plate for each experiment. Include various drugs, peptides, or viruses in th....

Discussion

The development of a three-dimensional organ culture system utilizing alginate hydrogels represents a versatile method for analyzing many different tissue types from a wide variety of organisms. The use of 3D cultures can be extended to the fields of tissue engineering and regenerative medicine as it provides a scaffold on which cells can grow¹¹. Currently, our laboratory is undergoing initial studies using human ovarian and fallopian tube tissue; however, culture of human and non-human primate follicles has b.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Leibovitz L-15 medium	GIBCO, by Life Technologies	11415
αMEM medium	GIBCO, by Life Technologies	12000-022
Sodium alginate	NovaMatrix
Sodium cacodylate	Sigma-Aldrich	C0250
Polypropylene mesh fiber	McMaster-Carr		45" wide roll; 0.0041" opening
Lipofectamine 2000	Invitrogen	11668
Penicillin/streptomycin	GIBCO, by Life Technologies	15070-063
Alginate lyase	Sigma-Aldrich	A1603
A1603	Sigma-Aldrich	C9697	From Clostridium histolyticum
Bovine serum albumin	MP Biomedicals	103700
Fetal bovine serum	GIBCO, by Life Technologies	Gibco
ITS solution	Roche Group	11074547001	Insulin/transferrin/sodium selenite
Cytokeratin 8 antibody	Developmental Studies Hybridoma Bank	TROMA-1	Use at 1:200
DAPI	Vector Laboratories	H1200
Bromodeoxyuridine	Sigma-Aldrich	B50002-1G	Final concentration 10 μM