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/pubmed?term=((The+amazing+osteocyte%5BTitle%5D)+AND+%22Journal+of+Bone+and+Mineral+Research%22%5BJournal%5D)">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/pubmed?term=((The+osteocyte%3A+An+endocrine+cell+...+and+more%5BTitle%5D)+AND+%22Endocrine+Reviews%22%5BJournal%5D)">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/pubmed?term=((The+role+of+the+osteocyte+in+bone+and+nonbone+disease%5BTitle%5D)+AND+%22Endocrinology+and+Metabolism+Clinics+of+North+America%22%5BJournal%5D)">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/pubmed?term=((Cell+line+IDG-SW3+replicates+osteoblast-to-late-osteocyte+differentiation+in+vitro+and+accelerates+bone+formation+in+vivo%5BTitle%5D)+AND+%22Journal+of+Bone+and+Mineral+Research%22%5BJournal%5D)">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/pubmed?term=((Control+of+osteocyte+dendrite+formation+by+Sp7+and+its+target+gene+osteocrin%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Osteoid-mimicking+dense+collagen%2Fchitosan+hybrid+gels%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">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/pubmed?term=((Biomineralization+in+humans%3A+making+the+hard+choices+in+life%5BTitle%5D)+AND+%22Annual+Review+of+Genetics%22%5BJournal%5D)">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/pubmed?term=((Functional+structure+and+composition+of+the+extracellular+matrix%5BTitle%5D)+AND+%22Journal+of+Pathology%22%5BJournal%5D)">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/pubmed?term=((An+improved+fluorescence+assay+for+the+determination+of+lymphocyte-mediated+cytotoxicity+using+flow+cytometry%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">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/pubmed?term=((Hypomorphic+conditional+deletion+of+E11%2FPodoplanin+reveals+a+role+in+osteocyte+dendrite+elongation%5BTitle%5D)+AND+%22Journal+of+Cellular+Physiology%22%5BJournal%5D)">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/pubmed?term=((Loss+of+DMP1+causes+rickets+and+osteomalacia+and+identifies+a+role+for+osteocytes+in+mineral+metabolism%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">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/pubmed?term=((Osteocyte+control+of+bone+formation+via+sclerostin%2C+a+novel+BMP+antagonist%5BTitle%5D)+AND+%22EMBO+Journal%22%5BJournal%5D)">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/pubmed?term=((FGF-23+in+fibrous+dysplasia+of+bone+and+its+relationship+to+renal+phosphate+wasting%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">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/pubmed?term=((Dynamics+of+the+transition+from+osteoblast+to+osteocyte%5BTitle%5D)+AND+%22Annals+of+the+New+York+Academy+of+Sciences%22%5BJournal%5D)">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/pubmed?term=((Involvement+of+3D+osteoblast+migration+and+bone+apatite+during+in+vitro+early+osteocytogenesis%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">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/pubmed?term=((High+mineralization+capacity+of+IDG-SW3+cells+in+3D+collagen+hydrogel+for+bone+healing+in+estrogen-deficient+mice%5BTitle%5D)+AND+%22Frontiers+in+Bioengineering+and+Biotechnology%22%5BJournal%5D)">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><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&amp;nbsp;</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&amp;nbsp; Biotechnology, Shanghai, China</td><td>SKSI</td><td></td></tr><tr><td>laser confocal fluorescence microscopy</td><td>Carl&amp;nbsp;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&amp;nbsp;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>&amp;beta;-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 ...

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

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Bioengineering

1.1K Views.  Shanghai Jiao Tong University School of Medicine. We hebben een protocol uitgewerkt om een 3D-systeem op te zetten met behulp van extracellulaire metriek samen met collageen voor het analyseren van vorming en genexpressie door middel van osteogenese. Extracellulaire metriek kan de transparantie van de collageen-éénvormgel vergroten en het is geschikt voor het onderzoeken van de dendrietvorming voor beeldvormingstechnieken. Om te beginnen pipetteer je langzaam 0,9 milliliter basaalmembraanmatrix in een nieuwe centrifugebuis van 15 millilite...

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

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Cancer Research

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 ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Medicine

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

3D Models in Cancer Biology: Invasion Dynamics in Heterospheroids

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

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

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