IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix

Summary

Here, we present a protocol for culturing IDG-SW3 cells in a three-dimensional (3D) extracellular matrix.

Abstract

Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular matrix (osteoid) express the Pdpn gene to form cellular dendrites and transform into preosteocytes. Later, preosteocytes express the Dmp1 gene to promote matrix mineralization and thereby transform into mature osteocytes.This process is called osteocytogenesis. IDG-SW3 is a well-known cell line for in vitro studies of osteocytogenesis. Many previous methods have used collagen I as the main or the only component of the culturing matrix. However, in addition to collagen I, the osteoid also contains a ground substance, which is an important component in promoting cellular growth, adhesion, and migration. In addition, the matrix substance is transparent, which increases the transparency of the collagen I-formed gel and, thus, aids the exploration of dendrite formation through imaging techniques. Thus, this paper details a protocol to establish a 3D gel using an extracellular matrix along with collagen I for IDG-SW3 survival. In this work, dendrite formation and gene expression were analyzed during osteocytogenesis. After 7 days of osteogenic culture, an extensive dendrite network was clearly observed under a fluorescence confocal microscope. Real-time PCR showed that the mRNA levels of Pdpn and Dmp1 continually increased for 3 weeks. At week 4, the stereomicroscope revealed an opaque gel filled with mineral particles, consistent with the X-ray fluorescence (XRF) assay. These results indicate that this culture matrix successfully facilitates the transition from osteoblasts to mature osteocytes.

Introduction

Osteocytes are terminally differentiated cells derived from osteoblasts^1,2. Once the osteoblast is buried by the osteoid, it undergoes osteocytogenesis and expresses the Pdpn gene to form preosteocytes, the Dmp1^{gene to mineralize the osteoid, and the} Sost and Fgf23 genes to function as a mature osteocyte in bone tissue³. Here, a 3D culturing system is introduced to identify dendrite extension and marker gene expression in the osteocytogenesis process.

IDG-SW3 cells are an immortalized primary cell line derived fr....

Protocol

This protocol is suitable for culturing cells in four wells of 24-well plates. If preparing multiple samples or plates, the amounts of the reagents should be increased accordingly.

1. Preparation of the collagen I mixture

NOTE: Collagen I and basement membrane matrix gel quickly at room temperature. Therefore, collagen should be handled on ice (2 °C to 8 °C). All the tips and tubes used must be prechilled unless otherwise indicated. All the .......

Discussion

A critical point in this protocol is that steps 1 and step 2 must be performed on ice to prevent spontaneous coagulation. In this method, the final concentration of collagen I was 1.2 mg/mL. Thus, an optimal ddH₂O volume should be calculated to match the different collagens from various manufacturers.

In vivo, osteocytogenesis involves a polar movement of osteoblasts from the surface to the interior of trabecular bone¹⁴. This protocol frees cells fro.......

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-ethylenediaminetetraacetic acid	Hyclone	SH30042.01
15 mL tubes	Corning, NY, USA	430791
7.5% (w/v) Sodium bicarbonate	Sigma-Aldrich, MO, USA	S8761
ascorbic acid	Sigma-Aldrich, MO, USA	A4544
Collagen I	Thermo Fisher Scientific	A10483-01
fetal bovine serum	Thermo Fisher Scientific	10099141
homogenizer	BiHeng  Biotechnology, Shanghai, China	SKSI
laser confocal fluorescence microscopy	Carl Zeiss, Oberkochen, Germany	LSM 800
Live/Dead Cell Imaging kit	Thermo Fisher Scientific	R37601
Matrigel matrix	Corning, NY, USA	356234
MEM (10X), no glutamine	Thermo Fisher Scientific	21430079
paraformaldehyde	Sigma-Aldrich, MO, USA	158127
phosphate buffered saline	Hyclone	SH30256.FS
stereo microscope	Carl Zeiss, Oberkochen, Germany	Zeiss Axio ZOOM.V16
Trizol	Thermo Fisher Scientific	15596026
X-ray fluorescence	EDAX, USA	EAGLE III
β-glycerophosphate	Sigma-Aldrich, MO, USA	G9422