We thank Dr. Lynda F. Bonewald for gifting the IDG-SW3 cell line. This work was supported by the National Natural Science Foundation of China (82070902, 82100935, and 81700778) and the Shanghai &#34;Science and Technology Innovation&#34; Sailing Project (21YF1442000).

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 &#176;C to 8 &#176;C). All the tips and tubes used must be prechilled unless otherwise indicated. All the procedures should be performed in a safety hood.
<ol>
	<li>Place all the reagents and centrifuge tubes on ice.</li>
	<li>Slowly pipette 0.48 mL of collagen I (5 mg/mL) and 0.1 mL of 10x minimum essential medium (MEM) (with phenol red) into a sterile 15 mL centrifuge tube kept on ice. Mix well by pipetting up and down gently.</li>
	<li>According to the color palette of the phenol red indicator, use an appropriate volume of 7.5% (w/v) NaHCO3 (approximately 0.2 mL) to adjust the phenol red indicator to orange, which indicates that the pH value is in the range of 7.0-7.4. 
	NOTE: Depending on the pH of the medium, a volume of NaHCO3 is used to bring the pH to approximately 7.0-7.4. Additionally, NaOH is used for pH neutralization.</li>
	<li>Bring the final volume of the mixture up to 1 mL by adding an appropriate volume of ddH2O. Mix well by pipetting gently up and down to prepare for use. 
	&#8203;NOTE: The final volume of ddH2O will depend on the amount of NaHCO3 or NaOH used in step 1.3.</li>
</ol>
2. Preparation of the cell-matrix mixture
<ol>
	<li>Slowly pipette 0.9 mL of basement membrane matrix into a new 15 mL centrifuge tube on the ice.</li>
	<li>Culture the IDG-SW3 cells in a T25 flask with 4 mL of complete medium (alpha-MEM containing 10% fetal bovine serum [FBS]) supplemented with 50 U/mL INF-&#947; at 33 &#176;C.</li>
	<li>Once the IDG-SW3 cells reach 90% confluency, remove the medium, and wash the cells with phosphate-buffered saline (PBS, pH 7.4 throughout the protocol) with a pipette. Digest the cells with 0.5 mL of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) at 37 &#176;C for 30 s.</li>
	<li>Inactivate the trypsin by adding 3.5 mL of complete medium (alpha-MEM containing 10% fetal bovine serum [FBS]). Transfer the 4 mL cell suspension to a 15 mL centrifuge tube.</li>
	<li>Spin down the cell suspension at 300 &#215; g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the cell pellet in 1 mL of the complete medium on ice.</li>
	<li>Count the cells using a hemocytometer and adjust the final cell density with the complete medium to 4 &#215; 105 cells/mL. Add 0.1 mL of the prepared cell suspension to 0.9 mL of the extracellular matrix from step 2.1. Mix well by pipetting gently up and down. 
	NOTE: If a cell-free control is needed, add 0.1 mL of the complete medium to 0.9 mL of extracellular matrix as a negative control.</li>
</ol>
3. Plating the cell-gel mixture
<ol>
	<li>Gently, mix well the two mixtures from step 2.6 and step 1.4 to 2 mL by pipetting up and down on the ice. Pipette 0.5 mL of the final mixture into each well (four wells of a 24-well plate). Incubate at 37 &#176;C for 1 h to form a cell-gel mixture.</li>
	<li>Add 0.5 mL of osteogenic medium (complete medium containing 50 &#181;g/mL ascorbic acid and 4 mM &#946;-glycerophosphate, also known as osteogenic induction medium)4 to the cell-gel mixture in each well to start the osteogenic differentiation at 37 &#176;C. Consider this as Day 0.</li>
	<li>Every 2 days, replace half the medium with fresh osteogenic medium maintained at 37 &#176;C. Continue the culturing for 35 days.</li>
</ol>
4. Identifying the cell viability and cellular dendrites using a confocal microscope
<ol>
	<li>Cell staining 
	NOTE: Calcein acetoxymethyl ester (calcein AM) is a cell-permeant dye that can be used to determine cell viability. Ethidium homodimer-I (EthD-1) does not cross the membrane of intact/viable cells9. Thus, calcein AM/EthD-1 can be used to identify cell viability and cellular dendrites.
	<ol>
		<li>On Day 1 and Day 7 of the culture, remove the calcein AM stock solution (4 mM in DMSO) and EthD-1 stock solution (2 mM in DMSO/H2O 1:4 [v/v]) from the freezer, and allow them to warm to room temperature. 
		NOTE: The mineralized matrix has a strong autofluorescence signal, so the preosteocyte stage without Dmp1 expression within 7 days of culturing4 is more suitable for the observation of cell staining.</li>
		<li>Add 4 &#181;L of the 2 mM EthD-1 stock solution and 5 &#181;L of the 4 mM calcein AM stock solution to 2 mL of Dulbecco&#39;s phosphate-buffered saline (D-PBS), and mix well. This gives approximately 2 &#181;M calcein AM and 4 &#181;M EthD-1 as working solutions.</li>
		<li>Pipette 0.5 mL of the working solution into the wells in the 24-well plate. Incubate for 30-45 min at room temperature to stain the cell-gel matrix.</li>
		<li>After incubation, add approximately 0.5 mL of fresh D-PBS to a new 35 mm glass-bottom culture dish. Cover the dish to prevent contamination or drying of the samples.</li>
		<li>Using elbow-tipped forceps, carefully transfer the stained cell-gel matrix from step 4.1.3 into the 35 mm culture dish from step 4.1.4. Avoid damaging or shearing the cell-gel matrix.</li>
		<li>View the labeled cells under a laser confocal fluorescence microscope.</li>
	</ol>
	</li>
	<li>Microscope scanning set
	<ol>
		<li>Place the 35 mm dish on the stage. Select the regions of the cell-gel matrix to be scanned through the eyepiece using a 10x objective. Higher-magnification objectives are not recommended due to the size of the extended dendrites.</li>
		<li>Select the optical filters. Calcein can be viewed as green with a standard fluorescein bandpass filter, and EthD-1 can be viewed as red with filters for propidium iodide or Texas Red dye. Select the &#34;line mode&#34; for scanning and set the &#34;pinhole&#34; to 2 &#181;m.</li>
		<li>Select a data depth of 8 bit data depth and an image resolution of 1,024 pixels x 1,024 pixels.</li>
		<li>Image using sequential line scanning with the optimal laser and detector settings (keep a minimal laser energy to avoid potential photobleaching). 
		NOTE: The optimal detector setting depends on the final fluorescence signals.</li>
	</ol>
	</li>
	<li>Collecting a &#34;z-stack&#34; of images
	<ol>
		<li>According to the green channel signal, define the top and bottom positions of the cell-gel matrix to be scanned by focusing through the sample while continuously scanning, .</li>
		<li>Once the top and bottom of the sample have been specified, select the desired scanning frame. Start scanning. The sample will be scanned from the top to the bottom with the selected settings from step 4.2, generating a gallery of images.</li>
	</ol>
	</li>
	<li>Cell viability identification.
	<ol>
		<li>Select one slice from step 4.3. The green and red channels indicate live and dead cells, respectively.</li>
	</ol>
	</li>
	<li>Cell dendrite identification.
	<ol>
		<li>Select the single green channel to generate 3D reconstructions with the image acquisition software. The depth information is displayed as a series of rainbow pseudocolor images. The dendrites located at the bottom of the gel are displayed in red, whereas those at the top are displayed in blue.</li>
	</ol>
	</li>
</ol>
5. Identifying appearance using a stereomicroscope
<ol>
	<li>On Day 1, Day 7, Day 21, and Day 35 of the culture, remove the culturing medium, and wash the gels in the plate twice with D-PBS. Add 0.5 mL of 4% (v/v) paraformaldehyde in D-PBS to the well to fix the cell-gel matrix in the plate for 10 min at room temperature.</li>
	<li>Remove the paraformaldehyde in the well and wash the cell-gel matrix two more times with D-PBS in the plate. Leave 0.5 mL of D-PBS in each well for imaging.</li>
	<li>Place the plate on the stage and select the optimal position through the eyepiece using a 0.5x objective. Image the full-field view of the cell-gel matrix in the plate individually using &#34;automatic exposure&#34; under the bright field.</li>
</ol>
6. Identifying mineral deposition with an XRF assay
<ol>
	<li>On Day 35 of the culture, check whether the liquid nitrogen is sufficient. Start the XRF system. Open the sample room and transfer the gels with elbow-tipped forceps from the plate to the middle of the sample stage of the instrument. Close the sample room and wait for 30 min to allow the instrument to cool before use.</li>
	<li>Set the &#34;Exposure time&#34; to 35 ms, the &#34;Spectrum range&#34; to 0-40 keV, and the &#34;Electric current&#34; to 770 &#181;A for acquisition setup.</li>
	<li>Choose three to five scanning sites for analysis by moving the sample stage.</li>
	<li>Select the elements (Ca and P) for detection. Start the detection and export the results. 
	&#8203;NOTE: Ca and P are the most abundant elements in hydroxyapatite.</li>
</ol>
7. Identifying functional gene expression
<ol>
	<li>On Day 1, Day 7, Day 21, Day 28, and Day 35 of the culture, remove the culturing medium and wash the cell-gel matrix twice with ice-cold D-PBS in the plate.</li>
	<li>Transfer the gels with elbow-tipped forceps from the plate to a 2.0 mL tube (according to the culture time, the cell-gel matrix will gradually&#160;shrink to approximately 0.1 mL on Day 35). Make sure one gel is placed in one tube. Add 1 mL of phenol-chloroform and two sterile stainless-steel beating beads (4 mm, as a lysing matrix) into each tube. Place the tubes symmetrically into the prechilled sample slot of the mechanical beating homogenizer.</li>
	<li>Set the beating to a frequency of 55 Hz and an amplitude of 1 cm in each 1 min of mechanical beating with a 10 s pause, with six cycles in total. Start the bead-beating.</li>
	<li>Extract the total RNA from the cell-gel matrix using the phenol-chloroform-isoamylachohol method. Reverse-transcribe 2 &#181;g of total RNA to cDNA with random hexamer primers.</li>
	<li>Design primers targeting &#946;-actin, Pdpn, Dmp1, Sost,&#160;and Fgf23 for real-time PCR (Table 2). &#946;-Actin is the housekeeping gene used for normalization. The Pdpn10 and Dmp111 genes are mainly expressed in pre-osteocytes and mineralized osteocytes, whereas Sost12 and Fgf2313 are mainly expressed in mature osteocytes.</li>
	<li>Place the tube on a thermocycler with the heated lid set to 105 &#176;C and perform PCR amplification using the following PCR cycling settings: 95 &#176;C for 5 s and 60 &#176;C for 30 s for 35 cycles with an initial denaturation step at 95 &#176;C for 5 min and a final extension step at 60 &#176;C for 30 s. Analyze the fold changes with the &#916;&#916;Ct method.</li>
</ol>

<ol>
	<li>Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amazing+osteocyte.">The amazing osteocyte.</a> Journal of Bone and Mineral Research. 26 (2), 229-238 (2011).</li><li>Dallas, S. L., Prideaux, M., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osteocyte:+An+endocrine+cell+...+and+more.">The osteocyte: An endocrine cell ... and more.</a> Endocrine Reviews. 34 (5), 658-690 (2013).</li><li>Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+osteocyte+in+bone+and+nonbone+disease.">The role of the osteocyte in bone and nonbone disease.</a> Endocrinology and Metabolism Clinics of North America. 46 (1), 1-18 (2017).</li><li>Woo, S. M., Rosser, J., Dusevich, V., Kalajzic, I., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+line+IDG-SW3+replicates+osteoblast-to-late-osteocyte+differentiation+in+vitro+and+accelerates+bone+formation+in+vivo.">Cell line IDG-SW3 replicates osteoblast-to-late-osteocyte differentiation in vitro and accelerates bone formation in vivo.</a> Journal of Bone and Mineral Research. 26 (11), 2634-2646 (2011).</li><li>Wang, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+osteocyte+dendrite+formation+by+Sp7+and+its+target+gene+osteocrin.">Control of osteocyte dendrite formation by Sp7 and its target gene osteocrin.</a> Nature Communications. 12 (1), 6271 (2021).</li><li>Chicatun, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoid-mimicking+dense+collagen/chitosan+hybrid+gels.">Osteoid-mimicking dense collagen/chitosan hybrid gels.</a> Biomacromolecules. 12 (8), 2946-2956 (2011).</li><li>Kawasaki, K., Buchanan, A. V., Weiss, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomineralization+in+humans:+making+the+hard+choices+in+life.">Biomineralization in humans: making the hard choices in life.</a> Annual Review of Genetics. 43, 119-142 (2009).</li><li>Bosman, F. T., Stamenkovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+structure+and+composition+of+the+extracellular+matrix.">Functional structure and composition of the extracellular matrix.</a> Journal of Pathology. 200 (4), 423-428 (2003).</li><li>Papadopoulos, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+fluorescence+assay+for+the+determination+of+lymphocyte-mediated+cytotoxicity+using+flow+cytometry.">An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry.</a> Journal of Immunological Methods. 177 (1-2), 101-111 (1994).</li><li>Staines, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypomorphic+conditional+deletion+of+E11/Podoplanin+reveals+a+role+in+osteocyte+dendrite+elongation.">Hypomorphic conditional deletion of E11/Podoplanin reveals a role in osteocyte dendrite elongation.</a> Journal of Cellular Physiology. 232 (11), 3006-3019 (2017).</li><li>Feng, J. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+DMP1+causes+rickets+and+osteomalacia+and+identifies+a+role+for+osteocytes+in+mineral+metabolism.">Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism.</a> Nature Genetics. 38 (11), 1310-1315 (2006).</li><li>Winkler, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteocyte+control+of+bone+formation+via+sclerostin,+a+novel+BMP+antagonist.">Osteocyte control of bone formation via sclerostin, a novel BMP antagonist.</a> EMBO Journal. 22 (23), 6267-6276 (2003).</li><li>Riminucci, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-23+in+fibrous+dysplasia+of+bone+and+its+relationship+to+renal+phosphate+wasting.">FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting.</a> Journal of Clinical Investigation. 112 (5), 683-692 (2003).</li><li>Dallas, S. L., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+transition+from+osteoblast+to+osteocyte.">Dynamics of the transition from osteoblast to osteocyte.</a> Annals of the New York Academy of Sciences. 1192, 437-443 (2010).</li><li>Robin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+3D+osteoblast+migration+and+bone+apatite+during+in+vitro+early+osteocytogenesis.">Involvement of 3D osteoblast migration and bone apatite during in vitro early osteocytogenesis.</a> Bone. 88, 146-156 (2016).</li><li>Chen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+mineralization+capacity+of+IDG-SW3+cells+in+3D+collagen+hydrogel+for+bone+healing+in+estrogen-deficient+mice.">High mineralization capacity of IDG-SW3 cells in 3D collagen hydrogel for bone healing in estrogen-deficient mice.</a> Frontiers in Bioengineering and Biotechnology. 8, 864 (2020).</li></ol>

The authors have nothing to disclose.

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...

Osteocytes are terminally differentiated cells derived from osteoblasts1,2. Once the osteoblast is buried by the osteoid, it undergoes osteocytogenesis and expresses the Pdpn gene to form preosteocytes, the Dmp1gene 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 from transgenic mice and can expand or replicate osteoblast to late osteocyte differentiation when cultured in different media4. Compared with MLO-A5, MLO-Y4, and other cell lines, the expression profile of functional proteins, the ability to perform calcium salt deposition, and the responses to various hormones in IDG-SW3 cells are more likely to be the same as those of primary osteocytes in the bone tissue4.
Compared to 2D systems, 3D culturing systems are more capable of mimicking the in vivo cellular growth environment, including the nutrient gradient, low mechanical stiffness, and surrounding mechanical range (Table 1). Most of the previous methods for culturing osteoblastic cells in a 3D system used collagen I as the unique component in formulations4,5,6, because collagen I fibers serve as the site of calcium and phosphorus deposition. However, an indispensable constituent in the osteoid, the extracellular matrix, contains a large group of cellular factors that promote cellular growth, adhesion, and migration7,8 and is transparent and convenient for imaging observation. Thus, this protocol uses Matrigel (hereafter referred to as basement membrane matrix) as a secondary component for the osteocytogenesis study.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-ethylenediaminetetraacetic acid</td>
			<td>Hyclone</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Corning, NY, USA</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>7.5% (w/v) Sodium bicarbonate</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>S8761</td>
			<td></td>
		</tr>
		<tr>
			<td>ascorbic acid</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10483-01&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10099141</td>
			<td></td>
		</tr>
		<tr>
			<td>homogenizer</td>
			<td>BiHeng&#160; Biotechnology, Shanghai, China</td>
			<td>SKSI</td>
			<td></td>
		</tr>
		<tr>
			<td>laser confocal fluorescence microscopy</td>
			<td>Carl&#160;Zeiss, Oberkochen, Germany</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Cell Imaging kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>Corning, NY, USA</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM (10X), no glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>21430079</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Hyclone</td>
			<td>SH30256.FS</td>
			<td></td>
		</tr>
		<tr>
			<td>stereo microscope</td>
			<td>Carl&#160;Zeiss, Oberkochen, Germany</td>
			<td>Zeiss Axio ZOOM.V16</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray fluorescence</td>
			<td>EDAX, USA</td>
			<td>EAGLE III</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>G9422</td>
			<td></td>
		</tr>
	</tbody>
</table>

idg-sw3 cell culture in a three-dimensional extracellular matrix

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.

After live/dead cell staining, the cells were visualized using a confocal laser microscope. All the cells were calcein AM-positive (green color), and there were almost no EthD-1-positive cells (red color) in the field, indicating that the gel system made by this method is highly suitable for osteocytogenesis (Figure 1A, left). To better determine the spatial distribution of the cells, a pseudocolor image was chosen to display the cell dendrites at different depths of the gel; red shows the d...

Watch this Scientific Journal Video about IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix at JoVE.com

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

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

Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular ...

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999 Views.  Shanghai Jiao Tong University School of Medicine. Here, we present a protocol for culturing IDG-SW3 cells in a three-dimensional (3D) extracellular matrix. 

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...