The authors would like to thank and acknowledge the Medical Research Council for funding this research - MRC-DPFS grant code G1000788-98812.

1.Scaffold Fabrication and Sterilization
<ol>
	<li>Dissolve PCL in 1,1,1,3,3,3&#8211;hexafluoroisopropanol to give a 10% w/v concentration. As described in Bosworth et al.,9 electrospin the polymeric solution (parameters: 20 kV, 1 ml/hr, 20 cm) and collect aligned fibers on the edge of a rotating mandrel (600 rpm). With a scalpel remove the ribbon of collected fiber and then cut into shorter lengths - 3 cm (for troughs and rotary vessels) and 4 cm (for cell culture inserts) lengths.</li>
	<li>Using fine forceps submerge individual strips in distilled water and remove.</li>
	<li>Holding both ends between thumb and forefinger; manually twist the strip until it resembles thread.</li>
	<li>Briefly submerge this thread-like scaffold in distilled water and place on clean, non-fibrous card to dry.</li>
	<li>Once dry, place individually in clean microcentrifuge tubes and add 1 ml of 50% v/v ethanol in distilled water. Close the lids and leave for 24 hr. 
	Note: Perform the following steps under laminar flow:</li>
	<li>Place microcentrifuge tubes in a laminar flow cabinet and aspirate the 50% v/v solution. Replace with 1 ml 70% v/v ethanol in distilled water, close lids and leave for 24 hr.</li>
	<li>Repeat for 90% and 100% v/v ethanol in distilled water (1 ml volumes).</li>
	<li>Wash scaffolds twice with phosphate buffered saline solution (PBS), 24 hr&#160;per wash (2 x 1 ml).</li>
	<li>Remove PBS and replace with 1 ml cell culture media. Note: Scaffolds are ready for use post-24 hr.</li>
</ol>
2 . Determining Scaffold Surface Area and Number of Cells
<ol>
	<li>Using a light microscope and imaging software, measure the diameter of the electrospun yarn along its length to determine a mean value.</li>
	<li>Assume the yarn to be a cylindrical rod and approximate the surface area using:</li>
	<li>Where A = Surface area, r = radius and h = length 
	Note: The surface area was calculated to be 18,902,800 &#181;m2. Furthermore, it should be noted that the actual surface area will be greater than this calculation as the yarn is composed of hundreds of fine fibers, which will increase the surface area. Consequently a larger number of cells should be able to attach to the scaffold. However, this does not affect a direct comparison between test groups being made.</li>
	<li>Determine the maximum number of cells that could attach to the exposed scaffold surface by: 
	Note: Using a light microscope and imaging software, the diameter of human mesenchymal stem cells was determined to be 20 &#181;m (assuming cells are round), therefore in this case, number of cells = 60,200.</li>
</ol>
3. Scaffold Set-up &#8211; Cell Culture Inserts (Figure 1A)
<ol>
	<li>Under laminar flow, open the sterile 6-well cell culture inserts and separate the shorter rings with teeth from the wider ringed bodies.</li>
	<li>Take the ring with teeth pointing upwards. Drape one of the 4 cm scaffolds over the centre of the ring making sure it overlaps on both sides. Take the ringed body and position over the toothed ring and scaffold and push downwards making sure the scaffold stays in position and lies through the centre of the cell culture insert.</li>
	<li>Place the cell culture insert with scaffold into the well of a 6-well, low binding plate.</li>
	<li>Add 10 ml of culture media to the scaffold.</li>
</ol>
4. Scaffold Set-up &#8211; Trough (Figure 1B)
<ol>
	<li>Under laminar flow, place poly(tetrafluoroethylene) (PTFE) troughs into individual petri dishes and add 10 ml of culture media to the trough.</li>
	<li>Using forceps drape one of the 3 cm scaffolds into the trough, making sure its length lies parallel to the trough&#8217;s longer edge.</li>
</ol>
5. Scaffold Set-up &#8211; Bioreactor Vessel (Figure 1C)
<ol>
	<li>Under laminar flow, dispense 10 ml sterile PBS through the bioreactor vessel&#8217;s main port and leave for 10 min.</li>
	<li>Remove the PBS and replace with 8 ml culture media.</li>
	<li>Using forceps, insert one of the 3 cm scaffolds into the vessel via the main port.</li>
	<li>Close off the main port.</li>
</ol>
6. Cell Counting
<ol>
	<li>Culture human mesenchymal stem cells (hMSC) derived from bone marrow according to manufacturer protocol up to passage 4 prior to harvesting.</li>
	<li>Aspirate the media from a 75 cm2 flask containing hMSCs derived from bone marrow (passage 4, 80% confluency).</li>
	<li>Wash the cells with 10 ml sterile PBS and aspirate.</li>
	<li>Add 3 ml trypsin and incubate the flask at 37 &#176;C, 5% CO2 until the cells have dislodged from the flask surface.</li>
	<li>Add 7 ml culture media to inactivate the enzyme and transfer this total volume (10 ml) to a centrifuge tube.</li>
	<li>Resuspend this cell suspension by pipetting up and down several times to homogeneously disperse cells within the media and limit cell agglomerates (10 ml pipette).</li>
	<li>Remove 20 &#181;l of cell suspension and transfer to a haemocytometer.</li>
	<li>Place the haemocytometer under a light microscope and image at x10 objective.</li>
	<li>Focus the gridlines and count the number of cells in the 4 x 4 squares in each corner of the grid and which fall within the square and those that cross the right hand or bottom boundary line. Count for each set of 4 x 4 squares (4 counts per grid).</li>
	<li>Repeat the resuspension and cell count three times (steps 6.6-6.9).</li>
	<li>Calculate the average cell count and determine the volume of media required for cell resuspension (cell concentration 60,200 in 200 &#181;l). For example, determine the average cell count from the haemocytometer and then determine the overall average number of cells from the three separate counts. Multiply this by 1 x 104 to give number of cells per ml and then multiply by the total volume of cell suspension to give total number of cells. Use the following equation to determine volume of media required for resuspension:</li>
	<li>Centrifuge the cell suspension at 241 x&#160;g for 5 min.</li>
</ol>
7. Cell Seeding
<ol>
	<li>Aspirate the media from the centrifuged tube leaving the cell pellet and replace with the calculated media volume.</li>
	<li>Resuspend the cells and media for an even mix.</li>
	<li>Using a P200 Gilson pipette, slowly dispense 200 &#181;l of cell suspension onto each scaffold by running the tip of the pipette along the scaffold length and below the media liquid surface. Leave undisturbed for 20 min.</li>
	<li>For bioreactor vessels, dispense 200 &#181;l of cell suspension through the main port. Top-up the remaining 2 ml culture media via the syringe ports to give a total volume of 10 ml.</li>
</ol>
8. Experimental Start
<ol>
	<li>Transfer the bioreactor vessels to the RCCS-4DQ bioreactor and set to rotate at 9 rpm.</li>
	<li>Transfer the well plates with cell culture inserts and troughs to the shaker plate and set to rotate at 30 rpm.</li>
	<li>Transfer the well plates with cell culture inserts and troughs to the shelf of a cell incubator set at 37 &#176;C, 5% CO2 (static culture).</li>
</ol>
9.&#160;DNA Assay
<ol>
	<li>Prepare solutions &#8211; lysis buffer, 1x TE buffer, DNA standards and cell DNA working solution - as per the manufacturer instructions.</li>
	<li>After 4 hr, remove the samples from the incubator and place under laminar flow.</li>
	<li>Remove the media fractions from all samples and place in separately labelled centrifuge tubes. Centrifuge the tubes 241 x g . Remove the supernatant and add 3 ml lysis buffer. Resuspend the solution to break-up the cell pellet.</li>
	<li>Using forceps remove the scaffolds and place in centrifuge tubes containing 3 ml lysis buffer (for the scaffolds within the bioreactor vessels, remove the scaffold prior to aspirating the media). For scaffolds held within cell culture inserts, first free the scaffold by cutting the scaffold close to the insert&#8217;s edge using a scalpel.</li>
	<li>Add 3 ml of lysis buffer to each scaffold receptacle and scrape the surface (vigorously agitate for the bioreactor vessels). Remove the lysis buffer and place in separately labelled centrifuge tubes.</li>
	<li>Vortex each centrifuge tube for approximately 1 min to ensure sufficient agitation of the cells and buffer and to encourage lysis of the cell membrane.</li>
	<li>In a black 96 well plate, add 100 &#181;l of lysis buffer for each sample fraction &#8211; scaffold, media and well (duplicate).</li>
	<li>In the dark, add 100 &#181;l of cell DNA solution to all wells containing lysis buffer and mix gently.</li>
	<li>Include wells with lysis buffer containing no DNA and cell DNA solution to provide a negative and positive control for the well plate.</li>
	<li>Using a fluorescence plate-reader, measure the absorbance of the wells using 485 nm excitation and 520 nm emission.</li>
	<li>Compare the data with the standard curve generated from the DNA standards as per the manufacturer instructions.</li>
</ol>
10. Scanning Electron Microscope (SEM) Fixation
<ol>
	<li>Perform the following steps under laminar flow: After 4 hr, remove all scaffolds from their receptacles and place within a new 6-well plate (separate wells).</li>
	<li>Wash the scaffolds twice with PBS.</li>
	<li>Perform the following steps on the open bench: To each well, add 2 ml of 1.5% v/v glutaraldehyde in PBS to ensure complete coverage of the scaffold.</li>
	<li>Leave the plate for minimum 30 min&#160;at 4 &#176;C for cell fixation.</li>
	<li>Remove the fixative solution and wash the scaffolds twice with PBS.</li>
	<li>Dehydrate the scaffolds with increasing concentrations of ethanol in distilled water starting with 50% v/v, followed by 70% v/v and 90% v/v. For each concentration, fully submerse the scaffolds in solution (2 ml) and leave for 3 min. Discard the solution and repeat.</li>
	<li>Dehydrate in 100% ethanol by fully submersing the scaffolds in solution (2 ml) and leaving for 5 min. Discard the solution and repeat.</li>
	<li>Chemically dry the scaffolds using hexamethyldisilazane (HMDS) within a fume cupboard. Immerse the scaffolds in HMDS (2 ml) and leave for 5 min. Remove the HMDS and repeat.</li>
	<li>Remove the HMDS and allow the scaffolds to dry. Mount the scaffolds on commercially available SEM stubs (in this case stainless steel stubs with adhesive carbon tabs).</li>
	<li>To ease viewing within the SEM, coat the samples using a gold-sputter coater for 2 min&#160;to ensure a thin and even coverage.</li>
	<li>Place samples within the SEM and visualize the cell-seeded scaffolds using a 5 KeV electron beam.</li>
</ol>

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The authors declare that they have no competing financial interests.

Electrospun fiber matrices fabricated from biopolymers are regularly used to support cell attachment and proliferation for biomaterial and/or tissue engineering applications11,12. In these cases, the matrices are often thin sheets of fibers that easily cover the entire base of a cell culture well plate and thus are in complete contact with seeded cells which improves cell attachment. However, if the biomaterial scaffold does not fully cover the base of the well plate, there is a high chance that a large proportion of the seeded cells will not stay in contact with the scaffold and ultimately will not be able to attach. This study investigated several different methods for seeding cells onto scaffolds that do not cover the base of the well plate, in order to determine an optimized technique that could be recommended for future cell-based experiments.
Five different set-ups were investigated (Figure 1): scaffolds (electrospun yarn) held using cell culture inserts within low binding well plates and either kept under static conditions or shaken at 30 rpm; scaffolds placed within narrow PTFE troughs and held static or shaken at 30 rpm; and scaffolds housed inside bioreactor vessels rotating at 9 rpm. Determining the number of cells that had adhered to the electrospun yarns by DNA assay demonstrated a low percentage of attachment for all seeding set-ups (Figure 2); and this was further confirmed from scanning electron micrographs (SEM) (Figure 3). Greatest cell attachment &#8211; 30% or ~18,060 cells -was observed for yarns that were held within cell culture inserts and subjected to continuous motion. Interestingly, lowest cell attachment (15%) was achieved for yarns held by cell culture inserts but kept under static conditions, which would suggest that the inclusion of radial motion has a positive effect on keeping cells in contact with the scaffold. However, it should be noted that continuous circling of the media&#8217;s flow might be responsible for the cell agglomerates observed from the SEM images. The shaker plate was set on its lowest setting &#8211; 30 rpm &#8211; which could be a limitation to this set-up. Using a slower radial motion may help to prevent or reduce cell agglomeration and could also improve cell attachment as cells will experience less force. Future experiments should focus on optimising the ideal shaker speed for improved cell attachment. Incorporating motion for yarns held within the troughs did not result in a similar trend, with both scenarios yielding 18% attachment (~10,836 cells); though this may be due to the partial floatation of the scaffolds within the troughs (observed for troughs placed on the shaker plate) as they were not anchored to the base. Partial floating of the scaffold will prevent any cells that have sunk to the bottom of the trough from coming into contact with the material and adhering. For this particular set-up, the trough was housed within a petri dish and a total 10 ml volume of media added. The small dimensions of the trough means that the majority of the media is present within the petri dish and if there is any movement, cells may drift away from the trough into the petri dish and remain completely out of reach of the scaffold. To overcome these limitations, further experiments should include an extra step in the protocol, whereby the ends of the scaffolds are pinned to the base of the troughs using sterile fine-needles, as this should prevent their floatation and movement (particularly for scaffolds exposed to radial motion), which ultimately should lead to an increased number of cells attaching to the scaffold. 16% of cells had attached to the yarns present within the rotary vessels. Despite being a well-established technique for 3D culture, problems did arise with the removal of scaffolds from the vessels&#8217; main port, which may have resulted in loosely attached cells being lost. Vessels that can be fully opened would eliminate this problem; these are available to purchase, but are considerably more expensive than the disposable vessels used in this study.
This study demonstrates the current issues with seeding scaffolds that do not cover the entire base of standard cell culture well plates. Seeding a known cell number resulted in less than a third attaching to the scaffold, despite the scaffold&#8217;s surface area allowing for all cells to adhere. This could have detrimental consequences in other cell-based assays that may assess the biocompatibility and cell-material / cell-cell behaviour and interactions with the scaffold as a potential future medical device. Further limitations of the study may include the 4 hr time-point &#8211; despite being long enough to ensure initial cell seeding (cells have been shown to firmly attach to substrates within thirty minutes13,14,15), it may be reasonable to investigate later time-points providing cells do not proliferate during a longer time-frame as this would otherwise skew the starting cell number. Reducing the volume of media, in this case 10 ml, could also improve contact between the cells and scaffold and ultimately increase cell attachment. Future studies should also consider cell viability as the process of cell seeding can cause cell damage and/or cell death16. Cell DNA assays do not differentiate between viable and non-viable cells, as such a live/dead assay, for example, would highlight the level of viability.
This investigation raises awareness to the actual number of cells that attach to the scaffold despite seeding a known quantity. For studies that rely on the starting number of cells, it is highly important that researchers know exactly how many of that figure do in fact adhere to the substrate of interest.

Scaffolds are routinely being developed and researched for biomaterial and tissue-engineering applications. As such, they are commonly seeded with cells and their in vitro behavior characterized via assays that determine cell proliferation and cell number, for example. For experiments such as these, it is imperative that the initial cell number is known and researchers often state the seeding concentration in terms of number of cells per ml or cm2. While this is good practice, especially for scale-up purposes, it does not account for the actual number of cells that adhere to the scaffold surface (which is also dependent on the adhesive properties of the biomaterial surface1). This is especially true for scaffolds that do not cover the entire base of the cell culture well plate as cells could fall away from the construct and, due to the often static nature of the experiment, may never come back into contact with the material of interest. Electrospun fiber yarns are a good example of a scaffold that does not cover the base of the well (Figure 1A). In this case, low binding well plates that have not been surface-treated should be used to prevent cells from attaching to the plate&#8217;s surface and hence distorting the results of any well-based assay.
Well plates are readily used for cell seeding onto scaffolds, but they are not the only method available. Rotary cell culture systems, a type of bioreactor developed by the Life Sciences Division at NASA in the late 1980&#8217;s, can similarly be used to seed scaffolds within a three-dimensional (3D) environment with simulated microgravity. This type of bioreactor remains a popular choice with researchers worldwide and has been incorporated in studies for cell signalling2,3, stem cells4,5 and tissue engineering6,7. What makes the rotary bioreactor preferable to well plates is the maintenance of a 3D environment, which helps to prevent differentiated cells from dedifferentiating, as is often the case when cultured within conventional 2D conditions8.
This paper investigates different techniques for seeding human mesenchymal stem cells on electrospun poly(&#949;-caprolactone) fiber yarns as fabricated in Bosworth et al.,9 in order to maximize the initial number of cells attaching to these scaffolds within a 4 hr period. For 2D culture, yarns were securely held within well plates or custom-made poly(tetrafluoroethylene) (PTFE) troughs and kept under static conditions, or shaken at 30 rpm. For 3D culture, yarns and cells were held within bioreactor vessels rotating at 9 rpm.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Distilled water</td>
			<td>in-house supply</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>1117271000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline solution</td>
			<td>Life Technologies</td>
			<td>70013016</td>
			<td></td>
		</tr>
		<tr>
			<td>Human mesenchymal stem cells</td>
			<td>PromoCell GmbH</td>
			<td>C-12974</td>
			<td></td>
		</tr>
		<tr>
			<td>MSC culture media</td>
			<td>PromoCell GmbH</td>
			<td>C-28010B</td>
			<td>Warmed to 37 oC before use</td>
		</tr>
		<tr>
			<td>Supplement mix</td>
			<td>PromoCell GmbH</td>
			<td>C-39810</td>
			<td>Add to culture media</td>
		</tr>
		<tr>
			<td>Antibiotic/antimyotic mix</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>Add to culture media</td>
		</tr>
		<tr>
			<td>Trypsin (0.05 %) EDTA (0.02 %)</td>
			<td>Sigma-Aldrich</td>
			<td>59417C</td>
			<td>Warmed to 37 oC before use</td>
		</tr>
		<tr>
			<td>Cell culture flasks (T75)</td>
			<td>Becton Dickinson Ltd</td>
			<td>353110</td>
			<td></td>
		</tr>
		<tr>
			<td>Low binding 6-well plates</td>
			<td>Costar Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well CellCrowns</td>
			<td>Scaffdex</td>
			<td>C00003S</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri-dish 50 ml deep</td>
			<td>Sterilin</td>
			<td>124</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE troughs</td>
			<td>in-house production</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable RCCS vessels 10 ml</td>
			<td>Synthecon</td>
			<td>D-410</td>
			<td></td>
		</tr>
		<tr>
			<td>4 Vessel Rotary Cell Culture System bioreactor</td>
			<td>Synthecon</td>
			<td>RCCS-4DQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker plate</td>
			<td>Stuart</td>
			<td>SSM1</td>
			<td>Mini Orbital Shaker</td>
		</tr>
		<tr>
			<td>Haemocytometer</td>
			<td>Digital Bio</td>
			<td>DHC-F01</td>
			<td>Disposable C-Chip</td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Deltalab</td>
			<td>352096, 429901</td>
			<td>15 ml and 50 ml</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hettich</td>
			<td>Rotafix 32 A</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 3 ml</td>
			<td>Shield Medicare Ltd</td>
			<td>3039820</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Sterilin</td>
			<td>40305, 47310, 40125</td>
			<td>5, 10 and 25 ml</td>
		</tr>
		<tr>
			<td>Gilson pipettes</td>
			<td>SLS</td>
			<td>F144801, F144802, F123600, F123601, F123602</td>
			<td>P2 - P1000</td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>SLS</td>
			<td>PIP7852, PIP7834, PIP7840</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro test tube 1.5 ml</td>
			<td>Eppendorf</td>
			<td>30125.15</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoGreen Assay</td>
			<td>Invitrogen</td>
			<td>P7589</td>
			<td>Assay set-up in the dark</td>
		</tr>
		<tr>
			<td>Black 96-well plate</td>
			<td>Greiner Bio One</td>
			<td>655086</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent plate-reader</td>
			<td>BGM Labtech</td>
			<td>FLUOstar Optima</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde 25 %</td>
			<td>TAAB Laboratories</td>
			<td>G002</td>
			<td>Made to a concentration of 1.5 % v/v in PBS</td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Sigma</td>
			<td>999-97-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium stubs (SEM)</td>
			<td>Agar Scientific</td>
			<td>G301</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon tabs (SEM)</td>
			<td>Agar Scientific</td>
			<td>G3347N</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold sputter coater</td>
			<td>Edwards</td>
			<td>S150B</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope (SEM)</td>
			<td>Phenom World</td>
			<td>Phenom Pro</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimizing attachment of human mesenchymal stem cells on poly(&#949;-caprolactone) electrospun yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

The results highlight the location of cells following 4 hr post-seeding for each experimental set-up investigated. Figure 2A demonstrates the percentage of cells that have attached to the scaffold surface during this time. A conversion factor of 8.5 pg/cell was used to convert the measured DNA content into cell number and thus determine the percentage of cells10. For all seeding set-ups investigated, the percentage of cell attachment is relatively low, with greatest cell adherence (30%) for scaffolds held within cell culture inserts and shaken at 30 rpm (Insert Shaker). Lowest adherence (15%) was for scaffolds held within the cell culture inserts and kept under static conditions (Insert Static).
A large number of cells were present within the media fraction (Figure 2B), most notably for cell culture inserts held within low binding plates (Insert Static) and rotary vessels being 50% and 51% respectively. Scaffolds held within the troughs demonstrated a raised number of cells present within the holder itself &#8211; 48% of cells for Trough Shaker and 50% for Trough Static.
Scanning electron microscopy allowed a visual assessment of the cell-seeded scaffolds (Figure 3). Representative images highlighted a limited presence of cells on the fibrous surface, irrespective of seeding set-up. However, a greater number of cells and cell agglomerates were present on the scaffolds held within the cell culture inserts and shaken at 30 rpm (Insert Shaker).
<img alt="Figure 1" src="/files/ftp_upload/52135/52135fig1.jpg" /> 
Figure 1.&#160;Experimental Set-ups, where Electrospun Yarn is Held within; (A) cell culture inserts and low binding well plate; (B) poly(tetrafluoroethylene) (PTFE) trough and petri dish; and (C) bioreactor vessel. <a href="https://www.jove.com/files/ftp_upload/52135/52135fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52135/52135fig2.jpg" /> 
Figure 2.&#160;Location of Cells 4 hr&#160;Post-seeding onto PCL Electrospun Yarns using Different Seeding Set-ups. (A) Demonstrates the percentage of cells that have attached to the PCL scaffold (mean &#177; standard deviation); (B) highlights the percentage spread of cell location within the three fractions &#8211; media, well and scaffold (n = 4, data presented as mean values). <a href="https://www.jove.com/files/ftp_upload/52135/52135fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52135/52135fig3.jpg" />
Figure 3.&#160;Representative Scanning Electron Micrographs for PCL Electrospun Yarns with Human Mesenchymal Stem Cells, 4 hr after Initial Seeding using Different Experimental Set-ups. (All images at 1,000X&#160;magnification, scale bar = 130 &#181;m.) <a href="https://www.jove.com/files/ftp_upload/52135/52135fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Optimizing Attachment of Human Mesenchymal Stem Cells on PolyÎµ-caprolactone Electrospun Yarns at JoVE.com

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

optimizing-attachment-human-mesenchymal-stem-cells-on-poly

Research

JoVE Journal

Bioengineering

9.6K Views.  The University of Manchester. This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Video: Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used techniques for the development of in vivo cancer models have considerable limitations. Therefore, in this study, we aim to implement cell sheet technology to develop an in vivo cancer model. Hepatocellular carcinoma (HCC) is successfully developed in nude rats using cell sheets created from HCC cell line cells. The cancer cell sheets are generated through intracellular adhesion and the formation of a stratified structure, controlled by the extracellular matrix. This allows for the HCC sheet transplantation into the liver and the creation of a tumor-bearing animal model within a month. In addition, the role of mesenchymal stem cells (MSC) in the development of this cancer model is investigated. In addition to the HCC cell line sheet, another two cell sheets are created: a sheet of HCC cells and bone marrow MSCs (BMMSCs) and a sheet of HCC cells and umbilical cord MSCs (UCMSCs). Sheets that have a combination of both HCC cells and MSCs are also capable of producing a tumor-bearing animal. However, the addition of MSCs reduces the size of the formed tumor, and this adverse effect on tumor development varies depending on the used MSCs' source. This indicates that a cell sheet made of certain MSC subtypes could be utilized in tumor management and control.

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

Here, we present a protocol to develop an in vivo cancer model using cell sheet technology. Such a model could be very useful for the evaluation of anticancer therapeutics.

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...