The study was supported by grants from the Fondazione Bambino Ges&#249; and Ricerca Corrente (Italian Ministry of Health) to C.C.&#160;&#160;We would like to thank Dr Enrico Bertini (Department of Neuroscience, Unit of Neuromuscular and Neurodegenerative Diseases, Laboratory of Molecular Medicine, Bambino Ges&#249; Children&#39;s Research Hospital), Dr Stefania Petrini (Confocal Microscopy Core Facility, Research Laboratories, Bambino Ges&#249; Children&#39;s Research Hospital), Giulia Pericoli&#160;(Department of Onco-hematology, Gene and Cell Therapy, Children&#8217;s Research Hospital Bambino Ges&#249;) and Roberta Ferretti (Department of Onco-hematology, Gene and Cell Therapy, Children&#8217;s Research Hospital Bambino Ges&#249;) for scientific discussions and technical help. Maria Vinci is recipient of a &#8220;Children with Cancer UK fellowship&#8221;.

1. Coating 96 well plates
NOTE: Different coatings were tested in the same plate but separate wells (see Supplemental File).
<ol>
	<li>Dilute the Matrigel 1:100 in DMEM. Add 100 &#181;L per well to the 96 well plates and incubate for 1 h at room temperature. Following this, remove the solution and wash the wells with 100 &#160;&#181;L&#160;of DMEM twice.</li>
	<li>Dilute laminin (20 &#181;g/mL, LN-521) in PBS (with calcium and magnesium). Add 100 &#181;L to the well and incu...

<ol>
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The use of iPSCs for disease modeling and future drug screening together with their possible application in precision medicine makes it a technology of great relevance and for this reason we believe that it is necessary to clearly understand the in vitro culturing condition that better resemble the physiological situation of embryonic stem cells. In this context, we tested different ECM coatings using&#160;wild type iPSCs in order to understand the conditions that allow the cells to remain in a healthy and undifferentiat...

Induced pluripotent stem cells (iPSCs) are obtained from somatic cells and can be differentiated into different cell types. They are often used as a system to model disease pathogenesis or perform drug screening, and also offer the potential to be used in the context of personalized medicine. Since iPSCs have great potential, it is important to fully characterize them for use as a reliable model system. We previously showed the importance of growing iPSCs in a hypoxic environment as these cells rely on glycolysis and an aerobic environment can cause redox imbalance1. iPSCs are also vulnerable to other culture conditions, particularly the extracellular environment. Optimization of culture conditions is a key issue to keep them healthy and proliferating. A healthy iPSC culture will lead to healthy differentiated cells that generally are the endpoint of the model used to understand molecular, cellular and functional features of specific human disorders or cellular processes.
In this study, a simple protocol has been used to test the confluence of iPSCs using different coating conditions in separate wells. iPSCs require a feeder layer of murine embryonic fibroblasts (MEF) in order to properly attach, but the coexistence of iPSCs and MEF makes it difficult to perform analysis like RNA or protein extraction since two populations of cells are present. In order to avoid the feeder layer, different proteins belonging to the extracellular matrix (ECM) have been used to recreate the natural cell niche and to have feeder free iPSC culture. In particular, Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, which is enriched in extracellular matrix proteins (i.e., laminin, collagen IV, heparan sulfate proteoglycans, entactin/nidogen, and growth factors)2,3. The other used coating conditions are instead purified proteins with known relevance in building the ECMs: laminin-521 is known to be secreted by human pluripotent stem cells (hPSCs) in the inner cell mass of the embryo and it is one of the most common laminins in the body after birth4,5,6,7,8,9,10,11; vitronectin is a xeno-free cell culture matrix known to support growth and differentiation of hPSC12,13,14,15,16; fibronectin is an ECM protein important for vertebrate development and the attachment and maintenance of embryonic stem cells in a pluripotent state17,18,19,20,21,22,23,24,25. Since different coating conditions are available, we compare them in terms of their effect on iPSCs&#8217; confluence.

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1123">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">10 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td style="width:281px;">Corning</td>
			<td style="width:77px;">4488</td>
			<td style="width:176px;">Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL high-clarity polypropylene (PP) conical centrifuge tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td>Tool</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (With Ca2+; Mg2+)</td>
			<td>Thermofisher</td>
			<td>14040133</td>
			<td>Medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (without Ca2+; Mg2+)</td>
			<td>Euroclone</td>
			<td>ECB4004L</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td>Corning</td>
			<td>4487</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture microplate, 96 WELL, PS, F-Bottom</td>
			<td>Greiner Bio One</td>
			<td>655090</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture plate, 6 well</td>
			<td>Costar</td>
			<td>3516</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM (Dulbecco&#39;s Modified Eagle&#39;s Medium- high glucose)</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>ED4SS-500g</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epi Episomal iPSC Reprogramming Kit</td>
			<td>Invitrogen</td>
			<td>A15960</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FAST - READ 102</td>
			<td>Biosigma</td>
			<td>BVS100</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Merck</td>
			<td>FC010</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>Reagent</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">H2O</td>
			<td></td>
			<td>MILLIQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst</td>
			<td>Thermofisher</td>
			<td>33342</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminin 521</td>
			<td>Stem Cell Technologies</td>
			<td>77003</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>LS25030081</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>Corning Matrigel hESC-Qualified Matrix</td>
			<td>354277</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">Mouse embryonic fibroblasts (MEF)</td>
			<td>Life Technologies</td>
			<td>A24903</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Medium</td>
			<td>Stem Cell Technologies</td>
			<td>85851</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Supplement</td>
			<td>Stem Cell Technologies</td>
			<td>85852</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vitronectin</td>
			<td>Stem Cell Technologies</td>
			<td>7180</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>Reagent</td>
		</tr>
	</tbody>
</table>

measuring the confluence of ipscs using an automated imaging system

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

In this study, we investigated iPSCs confluence when grown on different coating conditions. Using a cytometer, we were able to obtain readily informative results in triplicates in 5 days. Since iPSCs hardly attach to plastic vessels and a coating is necessary to support their proliferation, we decided to monitor the confluence of human iPSCs as it is indicative of the health of the cell culture and it may reflect on their differentiation potential. After in vitro expansion, we seeded the iPSCs on different ECM substrates...

Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

Measuring the Confluence of iPSCs Using an Automated Imaging System

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

This protocol is significant because it allows a quick assessment of induced pluripotent stem cell confluence on different extracellular matrix coating substrates in real time. The main advantage of this protocol is that it does not require the single cell suspension counting, thereby preventing growth perturbation to iPSCs, which are sensitive to passaging. Demonstrating the procedure will be Valentina Magliocca, a postdoc from my laboratory.To set up basement membrane matrix-coated plates, dilute basement membrane matrix, set a 1 to 100 ratio in DMEM, and add 100 microliters of the resulting solution to each well of a 96-well plate. When all of the wells have been coded, place the plate into the cell culture incubator for one hour. At the end of the incubation, discard the basement membrane matrix solution, and wash each well two times with 100 microliters of fresh DMEM per well.To set up laminin-coated plates, dilute 20 micrograms per milliliter of laminin in PBS with calcium and magnesium, and add 100 microliters of the laminin solution to each well of a new 96-well plate. Then incubate the plate at four degrees Celsius overnight before washing the wells two times with fresh DMEM as demonstrated. To set up Vitronectin-coated plates, dilute 10 micrograms per milliliter of Vitronectin in dilution buffer, and add 100 microliters of buffer to each well of a new 96-well plate.After one-hour incubation at room temperature, wash the wells with 100 microliters of PBS without calcium and magnesium per well. To prepare human fibronectin-coated plates, dilute 30 micrograms per milliliter of fibronectin in double-distilled water, and add 100 microliters of the fibronectin solution to each well of a new 96-well plate. After a 45-minute incubation at room temperature, wash the wells with fresh DMEM as demonstrated.Two days before setting up an iPSC culture, seed mouse embryonic fibroblasts at a 2.4 by 10 to the fourth per square centimeter density in complete cell culture medium into each of two wells of six-wells cell culture plates, and place the plates in the cell culture incubator. To set up an iPSC culture, warm a stock solution of cryopreserved iPSCs in a 37 degrees Celsius water bath. When the cells have just thawed, clean the vial with 70%ethanol, and place the cells into a biological safety cabinet.Using a 1000-microliter pipette, transfer the cells drop by drop into a sterile 15-milliliter conical tube containing five milliliters of pre-warmed cell culture medium. Collect the cells by centrifugation, and resuspend the pellet in four milliliters of fresh cell culture medium. Then plate 1 by 10 to the fifth cells into each of two wells of the mouse embryonic fibroblast-seeded six-well plates.After seeding, supplement the cell cultures with 10 micromolar of the ROCK inhibitor Y-27632, and place the plates in the cell culture incubator for four to five weeks. When the cells reach 70 to 80%confluency, treat each culture with one milliliter of 0.5 millimolar EDTA for three to five minutes at room temperature. When the cells have detached, split the cells at a one to four ratio in fresh cell culture medium, and plate the cells in each of the four remaining wells of the six-well plate under feeder-free conditions.Then return the plates to the cell culture incubator. To assess the effects of the different coatings on cell confluence after at least one month of culture under feeder-free conditions, use disposable counting slides to count the cells from each culture using a counting chamber. Dilute the cells to a one by 10 to the fourth cells per 200 microliters of medium concentration, and seed the cells in triplicate into each of three wells of one covering-coated 96-well plate per condition.When all of the cells have been plated, place the plates in the cell culture incubator for 24 hours. The next day and every day for the next five days thereafter, perform automated imaging of the cells using the auto-contrast and auto-exposure settings for optimal visualization. To evaluate the changes in focus due to the light refraction at the border of the wells, set the analysis settings to confluence analysis to apply a mask of 60, 80 or 100%per well, and use the different mask analysis settings to analyze the cell confluence at each time point.To compare the overall differences of the different coding conditions, use the sample data to perform the student's paired-sample T-test, reporting the quantitative results as the mean changes in confluence, plus or minus the standard error of the mean. For characterization of the cytoskeletal microfilaments, first, fix the cells with 100 microliters of 4%paraformaldehyde in PBS per well for 10 minutes at room temperature, followed by two 10-minute washes in 200 microliters of PBS per wash. Transfer the previously placed cover slip from the plate to the microscope slide.Add 100 microliters of 5%bovine serum and 0.1%Triton in PBS to each well for one hour at room temperature to block any non-specific binding, and wash the samples with two 10-minute PBS washes as demonstrated. After the second wash, add 100 microliters of Phalloidin conjugate working solution per sample for a one-hour incubation at room temperature, followed by two 10-minute washes in PBS. Next, stain the nuclei with Hoechst 33342 diluted to a one-to-10, 000 concentration in PBS for 10 minutes at room temperature followed by two PBS washes.After the second wash, rinse the cells with water, and let the plates dry under a chemical hood. When the glass coverslip has dried, cover the cells with 100 microliters of an appropriate mounting medium per well, and locate the cells on a laser scanning confocal microscope equipped with a white light laser source and a 405 nanometer diode laser at the appropriate excitation and emission wavelengths. Then select the 63x oil immersion objective, and acquire sequential confocal images of the cells.Phalloidin staining allows visualization of the degree of cell adhesion to the surface of a vessel and specifically, the coating used for the vessel. Cells that are adherent to a coating demonstrate clearly visible cytoskeletal microfilaments instead of collapsed microfilaments. Brightfield imaging also allows imaging of the level of adhesion of the iPSCs to the coated surfaces.iPSCs seeded on laminin demonstrate a high rate of cell proliferation in a linear fashion over time compared to other coatings. Cells seeded on basement membrane matrix, Vitronectin and human fibronectin, in contrast, exhibit a linear proliferation rate in the first 96 hours with an increased slope in the confluence curve during the last 24 hours independently of the mask used. As the initial difference at 24 hours for the different coatings can be due to differences in cell attachment, cell growth can be normalized to the 24 hour data for the later time points.As observed, no differences exist in terms of confluence among the different coatings, suggesting that the differences observed within the laminin-coated cultures are most likely due to an increased ability of the iPSCs to adhere to this coating when passaged. This study may contribute to the development of standardized methodologies for culturing iPSCs, and may aid in their future possible use in clinics. We are currently investigating the biology of the major cell surface receptors that mediate cell-ECM contacts and that may be responsible for the maintenance of their self-renewal ability.

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JoVE Journal

Biology

1.9K vues.  Ospedale Pediatrico Bambino Gesù, IRCCS. This protocol is significant because it allows a quick assessment of induced pluripotent stem cell confluence on different extracellular matrix coating substrates in real time. The main advantage of this protocol is that it does not require the single cell suspension counting, thereby preventing growth perturbation to iPSCs, which are sensitive to passaging. Demonstrating the procedure will be Valentina Magliocca, a postdoc from my laboratory.To set up basement membrane matrix-coated p...