Funding from the BBSRC is kindly acknowledged (grant number BB H011293/1).

Overview
Section 1 describes the preparation of pH responsive nanosensors and characterization of nanosensor response to pH using fluorescence spectrometry and their size using SEM.
Section 2 describes the preparation of electrospun polymer scaffolds and characterization of their morphology and size using SEM. Section 2 also describes the preparation of self-reporting scaffolds which are electrospun scaffolds with the inclusion of the pH responsive nanosensors. The resulting self-reporting scaffolds are characterized by SEM to assess whether any morphological changes in the electrospun fibers has occurred. The fluorescence from the self-reporting scaffolds resulting from the incorporated nanosensors is assessed by confocal microscopy.
Section 3 describes the culture of cells upon the electrospun scaffold and the self-reporting scaffold. The scaffolds are first sterilized in preparation for cell culture following which the scaffolds are assessed to determine whether sterilization and the culture of cells affect the fluorescence capability of the self-reporting scaffolds. The protocol also describes the delivery of nanosensors to cells that are cultured upon electrospun scaffolds. Lysotracker dye is used to ensure that nanosensors have not been taken into cellular acidic compartments.
1. Preparation and Analysis of pH Responsive Sol-gel Nanosensors
1.1 Preparation of pH Responsive Sol-gel Nanosensors
Note: This preparation should be performed in a fume hood.
<ol>
	<li>Prepare the fluorophores for use within the pH nanosensors by dissolving 5-(and-6)-carboxyfluorescein, succinimidyl ester (FAM-SE) (1.5 mg) in dimethylformamide (DMF) (1 ml) in a round bottomed flask and 6-carboxytetramethylrhodamine, succinimidyl ester (TAMRA-SE) (1.5 mg) in DMF (1 ml) in another round bottomed flask. Add 3-aminopropyltriethoxysilane (APTES) (1.5 mL) to each flask and stir under a dry nitrogen atmosphere (21 &deg;C) for 24 hr in the dark i.e. wrap the samples in foil.</li>
	<li>Add both of the above dyes (250 &#181;l) to a mixture of ethanol (6 ml) and ammonium hydroxide (30 wt %, 4 ml) contained in a round-bottomed flask and stir for 1 hr (21 &deg;C).</li>
	<li>Slowly add tetraethylorthosilicate (TEOS) (0.5 ml) to the mixture, continue stirring for a further 2 hr. The mixture will turn cloudy. The nanosensors can be collected by rotary evaporation. Nanosensors can be stored in a glass vial at 4 oC for future use. CAUTION: further handling of nanosensors in their dry form should be performed in a fume hood or in the case of weighing them the balance should be enclosed, for example in a Weighsafe.</li>
</ol>
1.2 Nanosensor Response to pH
<ol>
	<li>Prepare S&#248;rensen's phosphate buffer solutions ranging from pH 5.5-8.0 by mixing specific ratios of sodium phosphate monobasic (0.2 M) and sodium phosphate dibasic (0.2 M) stock solutions. Check the final pH using a pH meter. Make any minor adjustments to the pH using sodium hydroxide (NaOH) (4 M) or hydrochloric acid (HCl) (2 M).</li>
	<li>Suspend the pH nanosensors in the pH buffers (5 mg/ml) by firstly holding the vessel containing the nanosensor solution on a vortex for 3 min then submerging the vessel in an ultrasonicator until the solution becomes cloudy (approximately 5 min). Repeat these steps to fully suspend the nanosensors in solution.</li>
	<li>Place the first solution into an optical cuvette and use excitation wavelengths of 488 nm for 5-(and-6)-carboxyfluorescein (FAM) and 568 nm for 6-carboxytetramethylrhodamine (TAMRA). Collect the emission wavelengths at 500-530 nm for FAM and 558-580 nm for TAMRA using a fluorescence spectrometer.</li>
	<li>Change the solutions in the optical cuvette so that the pH can be observed at different pH and continue collecting the emission spectra.</li>
	<li>Calculate the ratio of the emission maxima produced at each pH value to produce a calibration curve.</li>
</ol>
1.3 SEM of Nanosensors
<ol>
	<li>Place a sample of the nanosensors onto a carbon coated electron microscope stub and sputter coat with gold for 5 min under an argon atmosphere.</li>
	<li>Observe by scanning electron microscopy (SEM). During imaging adjust the working distance, voltage and magnification to minimize electron charging (Figure 1).</li>
</ol>
2. Preparation and Analysis of PLGA Scaffolds
2.1 Electrospinning PLGA Scaffolds
<ol>
	<li>In a fume hood dissolve poly(lactic-co-glycolic acid) (PLGA) in dichloromethane (DCM) (20% (w/w)) with pyridinium formate (PF) (1% (w/w)). Place the solution into a 10 ml&#160;syringe with an 18 G blunt fill needle and securely fit to a syringe pump.</li>
	<li>Distance the needle tip 20 cm away from a 20 cm x 15 cm aluminum collecting plate.</li>
	<li>Attach the electrode of a high voltage power supply to the tip of the syringe and the earth to the aluminum collecting plate.
	 CAUTION: Care should be taken as high voltage is used i.e. always turn the electrical supply off before handling any of the electrospinning equipment.</li>
	<li>Deliver the solution using a constant flow rate of 3.5 m/hr at 12 kV for 2 hr. Electrospinning using these conditions will give a scaffold depth of approximately 60 &#181;m.</li>
	<li>Leave the scaffold in a fume hood for 24 hr to allow solvent residue to evaporate.</li>
</ol>
2.2 Electrospinning pH Responsive PLGA Scaffolds
<ol>
	<li>Prepare scaffolds incorporating pH responsive nanosensors as described in section 2.1 but with the modification of adding nanosensors to the polymer solution (5 mg/ml). Suspend the nanosensors in the PLGA solution with the assistance of ultrasonication (approximately 5 min) prior to placing into a 10 mL syringe.</li>
</ol>
2.3 SEM of PLGA Scaffold and pH Responsive Scaffold
<ol>
	<li>Place a sample of the PLGA scaffold or pH responsive scaffold onto a carbon coated electron microscope stub and proceed as described for SEM of nanosensors (Figure 1).</li>
</ol>
2.4 Calibration of pH Responsive Scaffold
<ol>
	<li>Place a sample of pH responsive scaffold into a 35 mm culture plate and immerse in appropriate buffer solutions.</li>
	<li>On a confocal microscope use a 488 nm argon laser to excite the FAM and a 568 nm krypton laser to excite the TAMRA within the nanosensors. Observe the fluorescence emission for pH responsive scaffolds at 500-530 nm for FAM and 558-580 nm for TAMRA. (Figure 2). Use sequential scanning to avoid collection of excitation wavelengths.</li>
</ol>
3. Cell Culture upon PLGA Scaffolds and pH Responsive PLGA Scaffolds
Note: The following should be performed in a cell culture cabinet.
3.1 Preparation of PLGA Scaffolds for Cell Culture
<ol>
	<li>Cut the PLGA or pH responsive PLGA scaffolds into 2 cm2 pieces.</li>
	<li>Sterilize scaffolds by irradiating with ultraviolet (UV) light at a distance of 8 cm for 15 min each side.</li>
	<li>Transfer the scaffolds to a 12-well culture plate and place a steel ring on top. Add a solution of Penicillin/Streptomycin (5% v/v) in phosphate buffered saline (PBS) to the inside and outside of the steel ring, incubate overnight (37 &deg;C, 5% CO2). Note the steel ring was manufactured at The University of Nottingham and has the following dimensions: 2 cm outer diameter, 1 cm inner diameter, 1 cm depth.</li>
	<li>Remove the sterilizing solution and wash with PBS.</li>
	<li>Add cell culture media to the inside (500 &#181;l) and outside (500 &#181;l) of the steel ring, place in an incubator (37 &deg;C, 5% CO2) whilst preparing a cell suspension.</li>
	<li>Prepare a cell suspension and perform a cell count using a Trypan Blue exclusion assay.</li>
	<li>Create a cell suspension to achieve a cell number of 1 x 106 cells/ml.</li>
	<li>Remove media from the inside and outside of the steel ring.</li>
	<li>Replace with prewarmed cell culture media on the outside of the steel ring (1 m).</li>
	<li>Add 300 &#181;l of the cell suspension to the inside of the steel ring - rock plate backwards and forwards gently to distribute cells.</li>
	<li>Place the cell culture plate into an incubator (37 &deg;C, 5% CO2) - cell attachment should have taken place within 24 hr, however continue culture for as long as the experiment requires - refreshing the media every 2-3 days.</li>
</ol>
3.2 Nanosensor Delivery to Cells Cultured upon PLGA Scaffold
<ol>
	<li>Seed cells onto PLGA scaffold as described in section 3.1. For this study a blank PLGA scaffold (not containing nanosensors) was used due to overlap of fluorescence emission when imaging.</li>
	<li>Place the cell seeded scaffold into an incubator (37 &deg;C, 5% CO2) for 72 hr to allow the cells to grow and migrate throughout the scaffold prior to nanosensor delivery.</li>
	<li>Prior to nanosensor delivery wash the cells with PBS and change the media from standard culture media to antibiotic and serum free media, incubate (37 &deg;C, 5% CO2) for 1 hr. Note: standard culture media for 3T3 cells comprises of Dulbecco's Modified Eagle's medium supplemented with fetal calf serum (10% (v/v)), L-glutamine solution (2 mM), penicillin/streptomycin (1% (v/v)).</li>
	<li>During the incubation period prepare the nanosensor-liposome complex by briefly sonicating nanosensors (5 mg) with Optimem (50 &#181;l) to create solution A.</li>
	<li>Create solution B by adding Lipofectamine 2000 (5 &#181;l) to Optimem (45 &#181;l), incubate at room temperature for 5 min.</li>
	<li>Add solution A to solution B, incubate at room temperature for 20 min to form solution C which is the nanosensor-liposome complex.</li>
	<li>Add solution C (100 &#181;l) to cells and incubate (37 &deg;C, 5% CO2) for 3 hr.</li>
	<li>Remove the cell seeded scaffolds from the incubator, aspirate media and wash twice with PBS before adding PBS (1 ml) to allow live cell visualization by confocal microscopy. Note: PBS was used in this protocol so that the phenol red indicator present in cell culture media did not cause autofluorescence and also because a calibration at different pH was being performed. However phenol red free media could be used for longer term analysis of cell cultures.</li>
	<li>Immerse the cell seeded scaffold into buffers of different pH and monitor the response of the nanosensors associated with the cells.</li>
	<li>Monitor the pHi by determining the ratio of the fluorescence intensities from FAM and TAMRA by observing fluorescence using confocal microscopy (Figure 3).</li>
</ol>
3.3 Nanosensor Location within Mammalian Cells
<ol>
	<li>Incubate cells with nanosensors containing only the FAM dye as described in section 3.2 (this is because the fluorescence emission of TAMRA is in the same spectral region as the LysoTracker Red). Prepare these nanosensors as described in section 1.1 but with the omission of preparing and adding TAMRA.</li>
	<li>Following the nanosensor delivery period dilute LysoTracker Red stock solution to 50 nM.</li>
	<li>Add an aliquot of the diluted solution (2 &#181;l) to cells cultured upon PLGA scaffolds.</li>
	<li>Incubate the cells with LysoTracker Red for 1 hr (37 &deg;C, 5% CO2).</li>
	<li>Following the incubation period remove the media and wash the cells twice with PBS (pH 7.4) before changing the media to PBS (pH 7.4) (2 ml) in preparation for viewing using confocal microscopy. Note: Phenol red free media could be used instead of PBS.</li>
	<li>Add the nuclear stain Draq5 to the PBS so the final concentration is 5 &#181;M and incubate with the cells for 3 min (37 &deg;C 5% CO2). It is not necessary to change the media following the incubation period with Draq5.</li>
	<li>Use a 488 nm argon laser to excite the FAM only nanosensors, a 568 nm krypton laser to excite the LysoTracker Red and 633 nm helium/neon laser to excite the Draq5 chromophore and observe fluorescence at 500-530 nm, 580-600 nm, 650-750 nm respectively (Figure 3). Use sequential scanning to avoid collection of excitation wavelengths.</li>
</ol>

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The authors declare that they have no competing financial interests.

Tissue engineering aspires to create biological substitutes that can be used both as in vivo like in vitro tissue models and in tissue replacement therapy to repair, replace, maintain or enhance the function of a particular tissue or organ. Synthetic substitutes have been used for many years to replace or aid repair of tissues but these often fail due to poor integration with the host tissue and/or infection, which ultimately leads to rejection or further revision surgery. Generating living tissue in th...

A key strategy in tissue engineering is the use of biocompatible materials to fabricate scaffolds whose morphology resembles the tissue that it is going to replace and is also capable of supporting cell growth and function1,2. The scaffold provides mechanical support by allowing cell attachment and proliferation yet allows cell migration throughout the interstices of a 3D cellular construct. The scaffold must also allow for the mass transport of cell nutrients and not inhibit removal of metabolic waste3.
Electrospinning has emerged as a promising method for the fabrication of polymeric scaffolds capable of supporting cellular growth4-6. The nonwoven electrospun fibers produced are suitable for cell growth as they are often porous and allow cell-cell interaction as well as cell migration throughout the interstices of a 3D cellular construct7. It is important to monitor cell viability during the period of culture and to ensure that cell viability is maintained throughout the whole of the 3D construct. For example, culture conditions such as oxygen and pH require careful control, as gradients in analyte concentration can exist within the 3D construct. Bioreactors or perfusion systems can be employed to mimic in&#160;vivo conditions of interstitial flow and as a result increase nutrient transfer and metabolic waste removal8. The question of whether such systems are ensuring constant microenvironmental conditions can be addressed by assessing the cellular microenvironment in real-time.
Key microenvironment metrics that could be monitored in real-time include: temperature, chemical composition of cell media, concentration of dissolved oxygen and carbon dioxide, pH, and humidity. Of these metrics, temperature can be most readily monitored using in situ probes. Methods for monitoring the remaining listed metrics commonly involve removal of an aliquot for sampling and therefore disturb the cell culture and increase the contamination risk. Continuous, real-time methods are being sought. Current monitoring methods usually rely upon instruments that physically probe the cellular construct such as a pH monitor or oxygen probe. However, these intrusive methods can damage the cellular construct and disturb the ongoing experiment. Noninvasive monitoring of analyte concentrations within the 3D construct could enable real-time monitoring of various environmental aspects such as nutrient depletion9. This would allow assessment of nutrient supply to deeper regions within the structure and determine whether metabolic waste was being removed effectively10,11. Systems that attempt to address the issue of invasiveness generally involve the use of a perfusion chamber that passes culture medium through both the culture vessel and to external sensors to monitor pH, oxygen and glucose12. There is increasing interest in developing sensors that can be directly integrated into the culture vessel that do not require removal of an aliquot for sampling and as such would provide in&#160;situ monitoring.
To address such shortcomings for in&#160;situ and noninvasive monitoring of microenvironmental conditions we have incorporated analyte responsive nanosensors into electrospun scaffolds to produce self-reporting scaffolds13. Scaffolds that act as sensing devices by monitoring fluorescence activity have been prepared previously, where the sensing device was either the actual polymeric scaffold created by electrospinning or through the use of an analyte responsive dye which is incorporated into the polymer prior to scaffold formation14,15. However, these sensing devices have the potential to give erroneous optical outputs caused by possible interference from other analytes. The use of a ratiometric sensing device such as those prepared in the described protocol holds the potential to eliminate these possible adverse effects and provide a response specific to the analyte in question.
The electrospun scaffolds presented here have been prepared from the synthetic co-polymer poly(lactic-co-glycolic acid) (PLGA), selected due to having Food and Drug Administration (FDA) approval, owing to its biodegradable and biocompatible properties and a track record of supporting the growth and function of various cell types16-18. The prepared ratiometric analyte responsive nanosensors are responsive to pH. The nanosensors incorporate two fluorescent dyes into a biocompatible sol-gel matrix where one dye, FAM is responsive to pH and the other, TAMRA acts as an internal standard as it is not responsive to pH. Furthermore the fluorescence of both FAM and TAMRA can be analyzed separately as they do not significantly overlap. Determining the ratio of the fluorescence emission of both dyes at specific wavelengths gives a pH response independent of other environmental conditions. The self-reporting scaffolds could allow repeat assessment of pH in situ and in real-time without disrupting the developed 3D model. We have demonstrated that these scaffolds are capable of supporting cell attachment and proliferation and remain responsive to the analyte in question. The kinetics of acidic by-products in engineered constructs remains understudied and as such using the pH responsive scaffolds could greatly facilitate such studies19. Furthermore, the use of the self-reporting scaffolds for tissue engineering applications presents the opportunity to fully understand, monitor and optimize the growth of 3D model tissue constructs in vitro, noninvasively and in real-time.
The pH responsive nanosensors have also been delivered to the intracellular environment of fibroblasts cultured upon electrospun PLGA scaffolds. The ratio of the fluorescence emission from the dyes were used to monitor pHi and compared to a self-reporting scaffold incorporating pH nanosensors. The delivery of nanosensors to cells cultured in a 3D environment could enable monitoring of analyte concentration deep within the construct in a nondestructive manner. Therefore nanosensors may be a viable imaging tool to nondestructively assess cell behavior throughout 3D constructs allowing long-term analysis. Screening the analyte concentration of individual cells within a 3D construct could ensure that they are receiving sufficient nutrient and oxygen concentrations. Monitoring process parameters could assist in the development of standardized techniques for the effective mass transport of oxygen and nutrients. The delivery of nanosensors to the intracellular environment and incorporation of nanosensors into polymeric scaffolds could be combined to allow assessment of cell viability as well as scaffold performance within 3D constructs during the tissue growth process. This may lead to increased knowledge of these constructs and progress the fabrication of biologically relevant tissue substitutes.

<table border="0" cellpadding="0" cellspacing="0" width="481">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Ethanol</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">32221</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Anhydrous dimethylformamide (DMF)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">270547</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">Ammonium hydroxide 50% (v/v) aqueous solution</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:137px;">35574</td>
			<td style="width:79px;">diluted to 30% (v/v) with pure water</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">TEOS</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">13190-3</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">3-Aminopropyltriethoxysilane (APTES)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">A3648</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">5-(and-6)-carboxyfluorescein, succinimidyl ester (FAM-SE)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">C1311</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">6-carboxytetramethylrhodamine, succinimidyl ester (TAMRA-SE)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">C1171</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Sodium phosphate monobasic (0.2 M)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">S-9638</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Sodium phosphate dibasic (0.2 M)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">S-0876</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">NaOH</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">S8045</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Trypsin/EDTA</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">T4174</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Penicillin/Streptomycin</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">P0781</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">PBS</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">D8537</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">DMEM</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">D6046</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">FBS</td>
			<td style="width:71px;">Source Bioscience</td>
			<td style="width:137px;">Batch-213-101992</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">L-Glutamine</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">G7513</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Lipofectamine 2000</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">11668-019</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Optimem</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">11058-021</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">LysoTracker Red</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">L-7528</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Draq5</td>
			<td style="width:71px;">Biostatus Ltd</td>
			<td style="width:137px;">DR50050</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Nitrogen gas</td>
			<td style="width:71px;">BOC</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">DCM</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">320269</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">TFA</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">T6508</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="211">
			<td height="211" style="height:211px;width:195px;">Confocal microscope</td>
			<td style="width:71px;">Leica TCS-SP</td>
			<td style="width:137px;"></td>
			<td style="width:79px;">equipped with argon and krypton lasers and a 63X 0.9NA water immersion lens</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">UV light</td>
			<td style="width:71px;">UVLS28 UVP, USA</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">Stirrer plate</td>
			<td style="width:71px;">SB161-3 Jencons-PLS</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">pH meter</td>
			<td style="width:71px;">Jenway model 3510</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">Rotary Evaporator</td>
			<td style="width:71px;">Buchi Rotary Evaporator R200</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Centrifuge (nanosensors)</td>
			<td style="width:71px;">Hermle Z300</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:195px;">Centrifuge (cell culture)</td>
			<td style="width:71px;">Thermo Scientific Heraeus Biofuge Primo</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">Vortex</td>
			<td style="width:71px;">Whirlimixer Fisherbrand</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">Ultrasonicator</td>
			<td style="width:71px;">FB11021 Fisherbrand</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">Aluminum sheet</td>
			<td style="width:71px;">Nottingham University</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">35mm cell culture plate</td>
			<td style="width:71px;">Iwaki</td>
			<td style="width:137px;">3000035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">10 ml syringe</td>
			<td style="width:71px;">Becton Dickenson</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;"></td>
			<td style="width:71px;"></td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:71px;"></td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">3T3 Fibroblast cells</td>
			<td style="width:71px;">European Collection of Cell Cultures</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">PLGA</td>
			<td style="width:71px;">Lakeshore Biomaterials</td>
			<td>7525 DLG 7E</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Pyridinium formate</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>P8535</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Trypan blue</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>T8154</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sodium phosphate monobasic</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>S9638</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Sodium phosphate dibasic</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>S5136</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">HCl</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>320331</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

self-reporting scaffolds for 3-dimensional cell culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

The size distribution of the prepared pH responsive nanosensors was characterized using SEM, where the population of nanosensors imaged were measured and found to have nanometer dimensions in the range of 240-470 nm (Figure 1A). The achievement of a narrow and reasonably small diameter is consistent with using the St&#246;ber method to prepare nanoparticles. It has been found that using a basic pH environment during the synthesis of nanoparticles i.e. nanoparticles prepared using the St&#246;ber...

Watch this Scientific Journal Video about Self-reporting Scaffolds for 3-Dimensional Cell Culture at JoVE.com

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

self-reporting-scaffolds-for-3-dimensional-cell-culture

Research

JoVE Journal

Bioengineering

13.1K Views.  University of Nottingham. Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.