Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Summary

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Abstract

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines¹ support our understanding of invasion of the uterine wall² and remodeling of uterine spiral arteries^3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts^5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation^7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.

The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies^10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu¹³.

We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues^7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models^10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Protocol

1. Collagen Bead Preparation

1. Prior to loading EVTs for 3-D cell culture, one needs to prepare the Cytodex-3 microcarrier beads:
   1. Weigh out the appropriate amount of Cytodex-3 beads required for the experiment. This protocol is adapted for the 10ml RCCS vessel, in which 0.05g of beads are needed. For a 50ml RCCS vessel, scale accordingly. In a 50mL autoclavable conical tube, mix 250 mg Cytodex-3 beads with 12mL Dulbecco phosphate buffered solution (DPBS). This amount is sufficient for 5 RCCS vessels.
2. Ensure adequate volume is present in the conical tube, as the autoclave process will result in evaporation. Loosely cap the ....

Discussion

The culture technique presented here provides investigators with highly invasive EVT-like cells. It has now been recognized that a loss of differentiation occurs in monolayers due to inhibition of cellular responses to chemical and molecular cues in three dimensions (apical, basal, and lateral cell surfaces)^10,13. This technique reflects characteristics noted in utero on invading EVT cells. As the procedure mimics conventional monolayer tissue culture time kinetics, but provides cells with differentia.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cytodex microcarrier beads	Sigma-Aldrich	C3275
Rotating Cell Culture System (RCCS)	Synthecon	RCCS-D	Includes rotor base, power supply, 4 disposable RCCS units
RCCS Disposable Units	Synthecon	Contact Synthecon
3ml Luer-Lock tip syringe	BD Biosciences	309585
10ml wide-tip serological pipette	BD Biosciences	357504
MEM Alpha	Invitrogen	12561-072
Leibovitz’s L-15 medium, powder	Invitrogen	41300-039
H2O, Endotoxin free	Fisher Scientific	MT-25-055-CM
Sodium Bicarbonate	Sigma-Aldrich	S-7795
Peptone	Fisher Scientific	BP1420-100
Fructose	Sigma-Aldrich	F3510-100
Galactose	Sigma-Aldrich	G5388-100
Glucose	Sigma-Aldrich	G7528-250
HEPES	Invitrogen	15630-080
L-Glutamine	Invitrogen	25030
Insulin-Transferrin-Sodium Selenite (ITS)	Sigma-Aldrich	I1884
FBS	Invitrogen	10437
Penicillin-Streptomycin	Invitrogen	15140