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.

Video: Self-reporting Scaffolds for 3-Dimensional Cell Culture

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...