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

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Biology

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Cancer Research

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo tissue repair. In these granular scaffolds, the innate porosity generated by the void space between microgels enables cell infiltration and migration. Controlling the void fraction and particle fraction is critical for MAP scaffold design, as porosity is a bioactive cue for cells. Spherical microgels can be generated on a microfluidic device for controlled size and shape and subsequently freeze-dried using methods that prevent fracturing of the polymer network. Upon rehydration, the lyophilized microgels lead to controlled particle fractions in MAP scaffolds. The implementation of these methods for microgel lyophilization has led to reproducible studies showing the effect of particle fraction on macromolecule diffusion and cell spreading. The following protocol will cover the fabrication, lyophilization, and rehydration of microgels for controlling particle fraction in MAP scaffolds, as well as annealing the microgels through bio-orthogonal crosslinking for 3D cell culture in vitro.

Minimizing the variability in the particle fraction within granular scaffolds facilitates reproducible experimentation. This work describes methods for generating granular scaffolds with controlled particle fractions for in vitro tissue engineering applications.

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo ...

Scalable Generation of Uniform Cell Spheroids Using a 3D Acoustic Assembly Device

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method for manufacturing high-quality and high-throughput cell spheroids using a 3D acoustic assembly device through maneuverable procedures. The acoustic assembly device consists of three lead zirconate titanate (PZT) transducers, each arranged in the X/Y/Z plane of a square polymethyl methacrylate (PMMA) chamber. This configuration enables the generation of a 3D dot-array pattern of levitated acoustic nodes (LANs) when three signals are applied. As a result, cells in the gelatin methacryloyl (GelMA) solution can be driven to the LANs, forming uniform cell aggregates in three dimensions. The GelMA solution is then UV-photocured and crosslinked to serve as a scaffold that supports the growth of cell aggregates. Finally, masses of matured spheroids are obtained and retrieved by subsequently dissolving the GelMA scaffolds under mild conditions. The proposed new 3D acoustic cell assembly device will enable the scale-up fabrication of cell spheroids, and even organoids, offering great potential technology in the biological field.

Author Spotlight: Development of a Scaffold-Free Acoustic Assembly Method for High-Quality 3D Cell Spheroid Culture 

Cell spheroids have been considered one potential model in the field of biological applications. This article describes protocols for scalably generating cell spheroids using a 3D acoustic assembly device, which provides an efficient method for the robust and rapid fabrication of uniform cell spheroids.

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Summary

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Chapters in this video

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production