We want to thank all the subjects involved in this study. We also appreciate the imaging assistance by Light Innovation Technology Ltd, Shenzhen. This study was supported by Natural Science Funding of China (Grant No. 82201851), Shenzhen Science and Technology Program (Grant No. JCYJ20210324141403009, RCYX20210609104608036), Shenzhen Key Medical Discipline Construction Fund (Grant No. SZXK028), and Shenzhen Baoan Women&#39;s and Children&#39;s Hospital (Grant No. BAFY 2023003).

NOTE: All reagents used in this protocol can be found in the Table of Materials. Unless otherwise specified, all media were pre-equilibrated to 37 &#176;C before use.
1. 3D&#160;printing of the scaffold and model construction
NOTE: The steps here were performed according to the manual book of the commercial 3D-metal printing machine. The steps are briefly described below (Supplementary File 1).
<ol>
	<li>Print bed leveling
	<ol>
		<li>Tap the Menu icon on the dashboard and navigate to Utilities. Select Bed Level to initiate the leveling routine.</li>
		<li>Remove the print sheet from the print bed and select Done. The print bed will ascend to its maximum height in preparation for leveling. Once the progress bar reaches 100%, press Start.</li>
		<li>If the Metal X printer has not been leveled previously, select Reset. If it has, choose Skip and proceed directly to Step 1.1.5 (the 16-point scan).</li>
		<li>Use a 2.5 mm hex key to turn all three-bed leveling screws clockwise until they are tight. Then, loosen them by turning counterclockwise two full rotations to set them at a mid-point height. Press Done to continue.</li>
		<li>The printer will scan the bed at 16 designated points. Refrain from touching the bed or frame during this process. Once the progress reaches 100%, press Next.</li>
	</ol>
	</li>
	<li>Print sheet application
	<ol>
		<li>Remove any residual metal or support debris. Toggle the Vacuum slider to On.</li>
		<li>Allow the bed to lower and heat up, then press Next. Place the print sheet over the vacuum grid and press Next.</li>
		<li>Position the sheet press over the print sheet, aligning it with the Z-rails at the rear of the chamber.</li>
		<li>Wait for the vacuum to engage and create a stable seal. Once the vacuum is engaged, press Done.</li>
	</ol>
	</li>
	<li>Metal filament loading
	<ol>
		<li>Manually position the print head in the center of the printing area. Remove the print head cover by sliding it upward and off the mounting screws.</li>
		<li>Tap the Menu icon on the option board. Select Materials from the options.</li>
		<li>Choose Load Metal to start the loading routine. Choose Quick Load.</li>
		<li>Select the specific type of metal material to be loaded (e.g., Aluminum 6061-T6 | 3.3211 | 65028 | AlMg1SiCu).</li>
		<li>Put the spool on the holder and press Next. Close the door and allow the spools to warm up, then press Next.</li>
		<li>Insert the material into the front inlet of the print head (labeled M) until the extruder begins to draw it through.</li>
	</ol>
	</li>
	<li>Metal part printing
	<ol>
		<li>In the Printing Settings panel, click Export Build. Save the file to a USB drive formatted to FAT32 and insert it into the printer.</li>
		<li>Select the Menu icon on the board. Navigate to Storage and select Print From Storage. Choose the part file to commence printing.</li>
	</ol>
	</li>
	<li>Metal part removing
	<ol>
		<li>Carefully slide the print sheet and printed parts off the print bed. Gently detach the parts from the print sheet. The flexible sheet should peel away with ease.</li>
	</ol>
	</li>
</ol>
2. Preparation for cell co-culture in the 3D model
NOTE: It is recommended that high-temperature resistant materials be used as consumables for 3D printing cell coverslip scaffold supports to facilitate autoclave sterilization before each use in cell culture experiments. Immortalized Human Endometrial Stromal Cells (HESC) and human endometrial epithelial cells (Ishikawa) were used in this study. The characterization of these cell lines has been previously reported5,6.
<ol>
	<li>Cell culture and passaging
	<ol>
		<li>Prepare 50 mL of complemental medium for hESC by adding DMEM/F12 medium + 2 mM L-Glutamine + 10% charcoal-stripped Fetal Bovine Serum (FBS) + 1% Penicillin/Streptomycin Solution.</li>
		<li>Prepare 50 mL of complemental medium for Ishikawa by adding Dulbecco&#39;s Modified Eagle Medium + 10% FBS + 1% Penicillin/Streptomycin Solution.</li>
		<li>Cell thawing 
		NOTE: The purchased cell line was shipped on dry ice in a frozen state. It should be stored in the vapor phase of liquid nitrogen or below -130 &#176;C.
		<ol>
			<li>Prepare all necessary sterilized equipment and dissociation enzyme, and warm the complemental medium to 37 &#176;C.</li>
			<li>Thaw the cells rapidly in a 37 &#176;C water bath with occasional gentle swaying. Transfer the cell suspension into a tube containing 5 mL of basal culture medium and centrifuge at 200 x g for 5 min.</li>
			<li>Carefully remove the supernatant. Resuspend the cell pellet in 5 mL of prepared complete culture medium and dispense it into two T25 culture flasks. Culture the cells in an incubator at 37 &#176;C and 5% CO2.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of cells with a coverslip
	<ol>
		<li>Use an 18 mm square coverslip as the surface of the cell culture; make sure the coverslip is clean and sterile.</li>
		<li>Coat the coverslip in the bottom of a 12-well plate with 200 &#181;L of extracellular matrix (ECM) at a concentration of 200-300 &#181;g/mL. Keep the plate at 37 &#176;C for 1 h.</li>
		<li>Remove the ECM from the plate. Inspect the cells for appropriate confluency, and make sure the density of both cell lines is around 70%-80%</li>
		<li>Remove the spent medium and wash the cells with 3 mL of phosphate-buffered saline (PBS) 2x.</li>
		<li>Add 2 mL of dissociation enzyme and culture the cells at 37 &#176;C for 2 min to detach them.</li>
		<li>Neutralize with complete culture medium and create a single-cell suspension by pipetting up and down.</li>
		<li>Take 100 &#181;L of the cell suspension and mix with trypan blue (dilution factor =2).</li>
		<li>Slide the coverslip over the chamber of the hemocytometer. Fill both side chambers with the cell suspension containing trypan blue.</li>
		<li>Count the number of cells in squares on both sides under a 20x microscope and calculate the density of cells.</li>
		<li>Transfer the cells to the prepared plate with fresh medium and incubate at 37 &#176;C with 5% CO2. Seed hESC into the well at 1 x 105 cells/mL for 24 h.</li>
		<li>For the Ishikawa cell line, seed the cell into the well without a coverslip the next day.</li>
	</ol>
	</li>
</ol>
3. Assembly of the co-culture model
<ol>
	<li>Soak the scaffolds with ethyl alcohol and double distilled water (ddw) thoroughly for 2 days. Sterilize scaffolds with an autoclave at 121 &#176;C and dry them.</li>
	<li>Add about 2 mL of complemental medium for the Ishikawa cell line to the well culturing Ishikawa, ensuring the liquid level covers the coverslip.</li>
	<li>Transfer the coverslip cover with hESC onto the scaffold and keep the side with the cell down. Plug the scaffold into the well-culturing Ishikawa. For the second set, invert this arrangement by transferring the coverslip cover with Ishikawa onto the scaffold and plugging the scaffold into the well-culturing hESC. Use the cells without the scaffold setup for independently growing cell growth analysis.</li>
	<li>Incubate the co-culture system at 37 &#176;C, 5% CO2. The co-culture period can vary depending on the experimental design but typically ranges from 24-72 h.</li>
</ol>
4. Image acquisition
<ol>
	<li>Carefully remove the culture medium and rinse cells gently with sterile phosphate-buffered saline (PBS).</li>
	<li>Remove coverslips with attached cells from culture dishes using fine forceps or tweezers. Optionally, place coverslips in a new dish with PBS to prevent drying during transfer.</li>
	<li>Take a clean microscope slide and place a drop of PBS. Gently transfer the coverslip containing cells onto the drop of mounting medium, ensuring cells face down onto the slide.</li>
	<li>Place the prepared slide onto the stage of the inverted microscope. Select the appropriate objective lens (e.g., 10x, 20x) and adjust focus using coarse and fine focus knobs.</li>
	<li>Set microscope parameters such as illumination intensity and camera settings. Using the microscope software, capture images of the cells at desired magnifications and fields of view. Adjust exposure time and other imaging settings to optimize image quality.</li>
</ol>
5. Image processing
<ol>
	<li>Click Open to open the image, click Process &#62; Filters &#62; Enhancement in order &#62; Choose Local Equalization, and click Apply.</li>
	<li>Click Edit &#62; Convert To &#62; Gray Scale 8. Click Enhance, choose Apply Contrast.</li>
	<li>Click Count and measure subjects. Click Manual, select a range, and adjust the histogram to make sure that all the cells are covered.</li>
	<li>Click Apply Mask, close the current window, Click Automatic Bright Objects &#62; Measure, Select Measurement, and choose Aera, Dendric, Density, and Size.</li>
	<li>Click Automatic Bright Objects, then click Count. Click Export Data to export the measurement data to a spreadsheet.</li>
</ol>
6. Cell harvesting
<ol>
	<li>Remove coverslips from the microscope slide and transfer them to a clean and dry well.</li>
	<li>Harvest the Ishikawa cell with RNA isolation reagent for transcriptomic or Ripa lysis reagent for proteomic research. 
	NOTE: For hESC, the coverslip-culture method was more effective for image capture after immune staining. Alternatively, the hESC could be harvested with RNA isolation reagent or Ripa lysis reagent. Cell lines cultured on the coverslip or the surface of the plate could be exchanged; however, we recommend that the cells prepared for imaging analysis be seeded on the coverslip.</li>
</ol>

3D-Printed Scaffold Model to Investigate Embryo Implantation Mechanisms

<ol>
	<li>Iske, J., Elkhal, A., Tullius, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fetal-maternal+immune+interface+in+uterus+transplantation.">The fetal-maternal immune interface in uterus transplantation.</a> Trend Immunol. 41 (3), 213-224 (2020).</li><li>Dey, S. 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=Molecular+cues+to+implantation.">Molecular cues to implantation.</a> Endocrine Rev. 25 (3), 341-373 (2004).</li><li>Li, X., 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=Three-dimensional+culture+models+of+human+endometrium+for+studying+trophoblast-endometrium+interaction+during+implantation.">Three-dimensional culture models of human endometrium for studying trophoblast-endometrium interaction during implantation.</a> Reprod Biol Endocrinol. 20 (1), 120 (2022).</li><li>Goss, 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=Mod3d:+A+low-cost,+flexible+modular+system+of+live-cell+microscopy+chambers+and+holders.">Mod3d: A low-cost, flexible modular system of live-cell microscopy chambers and holders.</a> PLoS One. 17 (6), e0269345 (2021).</li><li>Zheng, J., Liu, L., Wang, S., Huang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sumo-1+promotes+Ishikawa+cell+proliferation+and+apoptosis+in+endometrial+cancer+by+increasing+sumoylation+of+histone+h4.">Sumo-1 promotes Ishikawa cell proliferation and apoptosis in endometrial cancer by increasing sumoylation of histone h4.</a> Int J Gynecol Cancer. 25 (8), 1364-1368 (2015).</li><li>Damdimopoulou, P., 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=Human+embryonic+stem+cells.">Human embryonic stem cells.</a> Best Pract Res Clin Obst Gynaecol. 31, 2-12 (2016).</li><li>Gargett, C. E., Schwab, K. E., Deane, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+stem/progenitor+cells:+The+first+10+years.">Endometrial stem/progenitor cells: The first 10 years.</a> Hum Reprod Update. 22 (2), 137-163 (2015).</li><li>Lerman, M. J., Lembong, J., Gillen, G., Fisher, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3d+printing+in+cell+culture+systems+and+medical+applications.">3d printing in cell culture systems and medical applications.</a> Appl Phys Rev. 5 (4), 041109 (2018).</li><li>Palmara, G., Frascella, F., Roppolo, I., Chiappone, A., Chiad&#242;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+3d+printing:+Approaches+and+bioapplications.">Functional 3d printing: Approaches and bioapplications.</a> Biosens Bioelectron. 175, 112849 (2021).</li><li>Li, 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=Multiscale+architecture+design+of+3d+printed+biodegradable+zn-based+porous+scaffolds+for+immunomodulatory+osteogenesis.">Multiscale architecture design of 3d printed biodegradable zn-based porous scaffolds for immunomodulatory osteogenesis.</a> Nat Comm. 15 (1), 3131 (2024).</li><li>Arnold, J. T., Lessey, B. A., Sepp&#228;l&#228;, M., Kaufman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+normal+endometrial+stroma+on+growth+and+differentiation+in+Ishikawa+endometrial+adenocarcinoma+cells1.">Effect of normal endometrial stroma on growth and differentiation in Ishikawa endometrial adenocarcinoma cells1.</a> Cancer Res. 62 (1), 79-88 (2002).</li><li>Casper, R. F., Yanushpolsky, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+endometrial+preparation+for+frozen+embryo+transfer+cycles:+Window+of+implantation+and+progesterone+support.">Optimal endometrial preparation for frozen embryo transfer cycles: Window of implantation and progesterone support.</a> Fertil Steril. 105 (4), 867-872 (2016).</li><li>Cooke, P. 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=Stromal+estrogen+receptors+mediate+mitogenic+effects+of+estradiol+on+uterine+epithelium.">Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium.</a> Proc Natl Acad Sci U S Am. 94 (12), 6535-6540 (1997).</li><li>Owusu-Akyaw, A., Krishnamoorthy, K., Goldsmith, L. T., Morelli, S. S. <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+mesenchymal-epithelial+transition+in+endometrial+function.">The role of mesenchymal-epithelial transition in endometrial function.</a> Hum Reprod Update. 25 (1), 114-133 (2019).</li><li>Evans, J., Hutchison, J., Salamonsen, L. A., Azlan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+inflammasome+activation+accompanies+menstruation+and+may+have+implications+for+systemic+inflammatory+events+of+the+menstrual+cycle.">Endometrial inflammasome activation accompanies menstruation and may have implications for systemic inflammatory events of the menstrual cycle.</a> Hum Reprod. 35 (6), 1363-1376 (2020).</li><li>Go&#322;&#261;bek-Grenda, A., Olejnik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+modeling+of+endometriosis+and+endometriotic+microenvironment+-+challenges+and+recent+advances.">In vitro modeling of endometriosis and endometriotic microenvironment - challenges and recent advances.</a> Cellular Signal. 97, 110375 (2022).</li><li>Berger, C., Boggavarapu, N. R., Menezes, J., Lalitkumar, P. G. 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The authors declare that they have no conflicts of interest.

A simplified and cost-effective protocol is described for the indirect co-culture of endometrial stromal and epithelial cells. This method utilizes a homemade scaffold for cell slides, which comprises an upper support ring tailored for attachment to standard 12-well cell culture plates, complemented by a basal cell slide holder featuring four L-shaped rod-like structures. This setup facilitates the separation of endometrial stromal and epithelial cells while allowing for their interaction through the exchange of signalin...

Despite extensive research on human pregnancy, the molecular mechanisms at the maternal-fetal interface during implantation and early pregnancy remain poorly understood1. The human endometrium is mainly composed of two cell types: endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs). Implantation progresses through three stages: apposition, attachment, and invasion, which lead to the development of a competent embryo and receptive endometrium2. Considering the ethical constraints of in vivo studies on human subjects, as well as the difficulties in simulating the human condition in animals completely, the construction of in vitro human endometrium culture models has become an effective means of replicating the implantation and early pregnancy processes3. These models are valuable in investigating both normal and pathological pregnancies and provide a foundational platform for the preliminary testing and validation of therapeutic interventions in translational medicine.
Commercial chambers have been widely utilized in cellular biological research. These chambers offer valuable insights into cell migration and the crosstalk between different types of cells. However, commercial chambers are typically single-use and can be costly4.
A great number of in vitro human culture models consisting of endometrium and blastocyst, or blastocyst surrogates, have been developed to better understand the detailed process of implantation. These models, however, are still in their initial stage of application since the 3D structure is a highly complicated experimental setup, and certain specific differentiation media are expensive5,6,7.
The use of affordable 3D-printing hardware and a relatively short manufacturing period makes it possible to fit structures to target different experimental purposes. 3D printing techniques help reduce time costs and enable the creation of complex, customized structures. This technology significantly accelerates prototype design and iteration, making it a valuable tool for researchers across various fields, allowing them to complete their work more efficiently8,9,10.
Here, we present a feasible and economical experimental protocol for the construction of the 3D structure and its use as the cell culture system, which can simulate the interaction between endometrial stromal and epithelial cells for investigation of the endometrial receptivity during embryo implantation. This provides a customizable and low-cost alternative for commercial disposable material.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Hanks&#8242; Balanced Salt solution</td>
			<td>Solarbio</td>
			<td>H1046</td>
			<td>1/10</td>
		</tr>
		<tr>
			<td>12-well Clear TC-treated Plates</td>
			<td>Corning</td>
			<td>3513</td>
			<td>-</td>
		</tr>
		<tr>
			<td>25 cm&#178; Cell Culture Flask</td>
			<td>Corning</td>
			<td>430639</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Aluminum</td>
			<td>Markforged</td>
			<td>6061-T6</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Sigma-aldrich</td>
			<td>D2906</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium&#160;</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10099141C</td>
			<td>1/10</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10099141C</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS Premix</td>
			<td>Biocoat</td>
			<td>354350</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Corning</td>
			<td>354248</td>
			<td>ECM</td>
		</tr>
		<tr>
			<td>Metal X</td>
			<td>Markforged</td>
			<td>M F-PR-5000</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>Round Coverslip</td>
			<td>Biosharp</td>
			<td>BS-18-RC</td>
			<td>-</td>
		</tr>
		<tr>
			<td>TrypLE Select (10X)</td>
			<td>Gibco</td>
			<td>A1217701</td>
			<td>dissociation enzyme</td>
		</tr>
	</tbody>
</table>

co-culture model using two types of adherent cell lines

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications between various cell types within the decidua are crucial for regulating endometrial receptivity; however, the molecular mechanisms mediating this interaction are still unclear. In this regard, a model to study the implantation process is needed to establish a comprehensive in vitro model that can recapitulate the biology of endometrial epithelium-stroma interaction. This model is composed of regular cell-culture plates and a matching scaffold, which is generated by three-dimensional (3D) printing from low-cost materials. Here, we detail a set of protocols for model construction, cell preparation, cell seeding, cell culture, observation, and evaluation. Furthermore, we have included representative results with cells exhibiting good growth conditions under the microscope. This study aimed to develop in vitro models that would mimic the interaction between endometrial stromal cells and epithelial cells, as well as between trophoblast cells and endometrial cells.

Figure 1&#160;shows the homemade scaffold for cell slides used in this process, which comprises an upper support ring tailored for attachment to standard 12-well cell culture plates, complemented by a basal cell slide holder featuring four L-shaped rod-like structures.
According to Figure 2, the density of both cell types in the co-culture condition (Figure 2A,B) remained low after 72 h o...

Author Spotlight: In Vitro Endometrial Models to Study Epithelial-Stroma and Embryo Interactions

The protocol shows a novel in vitro experimental model that can recapitulate the biology of two kinds of adherent cell lines with a three-dimensional (3D)-printed scaffold. The construction of this model and operating procedures, from cell preparation and cell culture to analysis and evaluation, are described.

Co-culture Model Using Two Types of Adherent Cell Lines

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications ...

co-culture-model-using-two-types-of-adherent-cell-lines

Research

JoVE Journal

Biology

457 Views.  Shenzhen University. The protocol shows a novel in vitro experimental model that can recapitulate the biology of two kinds of adherent cell lines with a three-dimensional (3D)-printed scaffold. The construction of this model and operating procedures, from cell preparation and cell culture to analysis and evaluation, are described.

Video: Author Spotlight: In Vitro Endometrial Models to Study Epithelial-Stroma and Embryo Interactions

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

Barwy Pasażowanie ludzkich embrionalnych komórek macierzystych linii Trypsyna

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...