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

Summary

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.

Abstract

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.

Introduction

Microporous annealed particle (MAP) scaffolds are a subclass of granular materials in which the microgel (µgel) building blocks are interlinked to form a bulk, porous scaffold. With the unique microarchitecture of these granular scaffolds, the innate porosity generated by the void space between interlinked spherical microgel supports accelerated cell infiltration and migration¹. The microgel building blocks of MAP scaffolds can be fabricated from both synthetic and natural polymers with chemical modifications². The methods described here specifically highlight the use of microgels comprised of a hyaluronic acid (HA) backbone modified with functional norbornene (NB) handles. The NB functional handle on the HA polymer supports click chemistry reactions for forming microgels and linking them together to generate MAP scaffolds^3,4. Numerous schemes have been employed for linking the microgels together (i.e., annealing), such as enzymatic¹, light-based^5,6, and additive-free click chemistry^3,7 reactions. Additive-free click chemistry is described in this work, using the tetrazine-norbornene inverse electron demand Diels-Alder conjugation for interlinking the HA-NB microgels.

To fabricate MAP scaffolds, users first generate the microgel building blocks using reverse emulsions either in batch systems or within microfluidic devices, as well as with electrohydrodynamic spraying, lithography, or mechanical fragmentation². The production of spherical HA-NB microgels has been well described and previously reported using both batch emulsion² and microfluidic droplet generation techniques^8,9,10,11. In this work, spherical HA-NB microgels were generated on a flow-focusing microfluidic platform for controlled size and shape, as previously described^8,9,10. After purification, the microgels exist in an aqueous suspension and must be concentrated to induce a jammed state. When jammed, microgels exhibit shear-thinning properties, which allow them to function as injectable, space-filling materials¹. One method of inducing a jammed state is to dry the microgels via lyophilization, or freeze-drying, then subsequently rehydrate the dried product in a controlled volume¹². Alternatively, excess buffer can be removed from the microgel slurry via centrifugation over a strainer or with manual removal of the buffer from the microgel pellet either by aspiration or using an absorbent material. However, using centrifugation to dry the microgels can generate a highly variable range of particle fractions and void fractions when making granular scaffolds¹². Techniques for lyophilizing microgels have been described using 70% IPA for polyethylene glycol (PEG) microgels¹³, fluorinated oils for gelatin methacryloyl (GelMa) microgels¹⁴, and 70% ethanol for HA microgels¹². This protocol highlights methods for freeze-drying spherical HA microgels using 70% ethanol, a standard laboratory reagent, to retain the original microgel properties during the drying process. The freeze-dried HA microgels can be weighed and rehydrated at user-defined weight percentages to control the final particle fractions in MAP scaffolds¹².

The final step in MAP scaffold formation relies on annealing the microgels to create a bulk, porous scaffold¹. By utilizing native extracellular matrix components and employing bio-orthogonal annealing schemes, MAP scaffolds serve as a biocompatible platform for both in vitro cell culture and in vivo tissue repair³. Through these approaches, MAP scaffolds can be fabricated from HA-NB building blocks with user-defined particle fractions for their employment in tissue engineering applications¹². The following protocol describes the microfluidic production of HA-NB microgels followed by lyophilization and rehydration for controlling particle fraction in MAP scaffolds. Lastly, steps for annealing the microgels are described using bio-orthogonal chemistry for in vitro 3D cell culture experiments.

Protocol

1. Microfluidic device fabrication

1. Soft lithography
   NOTE: This protocol describes device fabrication of a flow-focusing microfluidic device design from de Wilson et al.⁹. However, this protocol can be used with any device design on an SU-8 wafer. The wafer can be taped to a Petri dish, and then needs to be silanized to prevent adherence of the PDMS to the wafer features¹⁵.
   1. Mix the polydimethylsiloxane (PDMS) elastomer base with the curing agent (see Table of Materials) at a 10:1 ratio. Prepare approximately 100 g to cover the wafer with ~5 mm PDMS. Pour the PDMS mixture onto the wafer and degas in a desiccator for approximately 30 min. Once all the bubbles are gone, place in an oven at 60 °C for at least 2 h to cure the PDMS.
   2. Use a knife to gently trace around the parameter of the device without cracking the wafer; then, carefully peel the PDMS off the wafer. Use a 1 mm biopsy punch (see Table of Materials) to create the inlet and outlet channels.
      NOTE: Be gentle when punching the microfluidic device. Tears or rips around the inlet or outlet channels can cause leaks during microgel production.
   3. Use tape to remove dust from the device on the feature side. Place the devices and clean glass slides on a hot plate at 135 °C for at least 15 min to remove moisture.
   4. In a fume hood, use a corona plasma gun (see Table of Materials) on high on both the glass slides and devices (feature side exposed) for approximately 30 s, and then quickly bond them together. Gently apply pressure to ensure a good seal between the device and the glass slide. Place the devices in a 60 °C oven overnight to secure the bond.

2. Microfluidic production of hyaluronic acid (HA) microgels with norbornene (NB) functional handles

1. HA-NB synthesis
   NOTE: HA-norbornene (HA-NB) synthesis was adapted from Darling et al.³ using 79 kDa sodium HA with molar equivalents of 1:1.5:2.5 of HA-repeat units to 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) to 5-norbornene-2-methylamine (NMA).
   1. Weigh the reactants. Dissolve the HA at 20 mg/mL in 200 mM MES buffer (pH ~6) by stirring in a beaker or flask on a stir plate. Once dissolved, add the DMTMM to the HA solution and allow to react for approximately 20 min at room temperature. For example, 1 g HA + 1.09 g DMTMM + 845 µL NMA can be used.
   2. Add NMA dropwise to the HA/DMTMM solution. Add parafilm to the opening of the reaction vessel to minimize evaporation and cover the reaction vessel with foil. Continue stirring while allowing the reaction to proceed for approximately 24 h.
   3. After 24 h, chill 200 proof ethanol (approximately 10x the reaction volume). On a stir plate, transfer the reaction dropwise to the chilled ethanol to precipitate the HA-NB and continue stirring at 200-300 rpm for 20 min.
   4. Transfer the solution to 50 mL conical tubes, and then centrifuge at 5,000 x g for 10 min. Pour off the excess ethanol to dispose as waste. At this point, the HA-NB product should be white pellets in the conical tubes. Pull vacuum on the HA-NB in a dessicator to dry overnight.
   5. Purify the HA-NB using 12-14 kDa molecular weight cut-off cellulose dialysis tubing (see Table of Materials). Dissolve HA-NB in 2 M NaCl solution and transfer to the dialysis tubing. Tie the tubing and secure with clamps, if needed. Transfer the filled dialysis tubing to a bucket with 5 L of ultrapure water and dialyze the HA-NB against water overnight.
   6. The next day, remove the water and replace with 1 M NaCl solution for 30 min. Remove the NaCl solution, and then dialyze against ultrapure water for 3 days, replacing the water daily.
   7. Filter the dialyzed product using 0.2 µm vacuum-driven filter, and then transfer the filtered product to 50 mL conical tubes.
   8. Add liquid nitrogen to a cryogenic container and flash-freeze the HA-NB tubes for 10 min. Then, remove the conical tubes with forceps and quickly remove the cap and cover with a lab-grade tissue (see Table of Materials). Secure the tissue with a rubber band and transfer to a lyophilization container or chamber (see Table of Materials) and lyophilize. Store the lyophilized product at -20 °C.
      CAUTION: Liquid nitrogen is a hazardous substance. Wear the appropriate personal protective equipment when working with liquid nitrogen.
   9. Quantify norbornene modification by dissolving the HA-NB at 10 mg/mL in D₂O and analyzing via proton NMR (Figure 1A)¹⁶.
      1. To determine the amount of functionalization, first calibrate the D₂O solvent peak to 4.8 PPM. Integrate the peak for the HA methyl protons (δ2.05) and calibrate the integration to 3.0. Next, integrate the peaks for the pendant norbornene groups at δ6.33 and δ6.02 (vinyl protons, endo). Normalize the integration of these peaks to the corresponding number of protons to determine the average degree of modification³.
2. Preparation of HA-NB microgel precursor
   1. Prepare 50 mM HEPES buffer (pH 7.5) and sterile filter the buffer using a 0.2 µm vacuum-driven filter. Using the HEPES buffer, prepare respective 50 mM stocks of lithium phenyl(2,4,6,-trimethylbenzoyl)phosphinate (LAP) photo-initiator and tris(2-carboxyethyl)phosphine (TCEP) reducing agent. Keep the LAP solution away from light.
   2. Prepare the other microgel precursor components by preparing respective 50 mM stocks of di-thiol linker and RGD peptide in sterile distilled water. Weigh out HA-NB and dissolve in HEPES buffer to prepare a 10 mg/mL stock.
      NOTE: Different di-thiol linkers could be used for the internal crosslinking of the microgels based on user preference. Both a degradable (i.e., MMP-cleavable) and non-degradable (dithiothreitol or DTT) linker have been listed in the Table of Materials. The RGD peptide is included in the microgel formulation to promote cell adhesion in MAP scaffolds, but this component could be removed and replaced with equal volume of HEPES buffer.
   3. Combine the precursor components with final concentrations of 9.9 mM LAP, 0.9375 mM TCEP (4 thiol/TCEP), 2.8 mM di-thiol linker, 1 mM RGD peptide, and 3.5 wt% (w/v) HA-NB by adding extra HEPES buffer to reach the desired final volume. Mix the precursor well using a positive displacement pipette.
   4. Using a P1000 pipette, slowly pull up the entire mixture. Put the tip onto the end of a 1 mL syringe and eject the tip from the pipette. Pull the syringe plunger to load the mixture into the syringe, and then add a 0.2 µm filter on the end of syringe and filter into a new 1.5 mL microcentrifuge tube. Centrifuge the filtered precursor solution to remove the bubbles produced during filtering.
   5. Again, using a P1000 pipette, slowly pull up the filtered precursor being careful not to create bubbles. If there are bubbles, gently tap the tip for them to dislodge and float to the top.
   6. Place the tip onto the end of a 1 mL syringe and eject the tip from the pipette. Keep the syringe vertical and pull the syringe plunger slowly until the entire precursor solution is in the syringe. Add a blunt tip needle to the syringe and push the precursor through the tip of the needle. Wrap the syringe in foil to keep out of light.
3. Preparation of microgel pinching solution
   1. Prepare 5% v/v Span-80 in heavy white mineral oil and mix well. Desiccate to remove bubbles. Keep the surfactant/oil mixture at room temperature wrapped in foil. Mix well and desiccate prior to each use.
   2. Use a 5 mL syringe to draw up the oil/surfactant mixture (minimize bubbles) until the distance between the plunger and the fingerhold is approximately equal to the distance of the precursor syringe. Add a blunt needle to the syringe and push the oil through the tip of the needle.
4. Microfluidic device setup
   1. Add a blunt needle to a 1 mL syringe and fill with synthetic hydrophobic treatment solution (see Table of Materials). Gently flow the solution through the microfluidic device until it pools at each inlet/outlet. Let the solution dry in the device on the benchtop for approximately 30 min, and then pull vacuum on the outlet to remove excess solution. Secure the device with clamps on a tabletop microscope.
   2. Wrap a 15 mL conical tube with foil and place in a tube rack to serve as the microgel collection container. Use a ring stand with a clamp to place the UV light probe into the opening of the collection tube. Use a UV detector (see Table of Materials) to measure the UV intensity, moving the probe until 20 mW/cm² is achieved. Turn off the UV light until later.
   3. Cut tubing at a length that will reach from the microfluidic device to the collection container. On one end of the tubing, cut a 45° angle. Gently insert the angled end of the tubing into the outlet channel.
      NOTE: Be gentle when inserting the tubing into the microfluidic device. Tears or rips around the inlet or outlet channels can cause leaks during microgel production.
   4. Secure both the precursor and oil phase syringes on a dual-syringe pump (see Table of Materials). Cut two more pieces of tubing at a length that will reach from syringe tips to the microfluidic device. On one end of each tube, cut a 45° angle. Carefully secure the tubing (blunt end) on both syringe tips.
   5. Change the settings on the pump for the 1 mL syringe and include the approximate precursor volume. Slowly push the pump forward until enough pressure is applied to the syringe plungers to push both the oil and the precursor to the ends of the tubing, removing any air from the system. Let the pressure equalize 5-10 min prior to moving on to step 2.4.6.
   6. Gently insert the angled end of the tubing into the inlet channels of the microfluidic device with the microgel precursor solution in the front inlet and the pinching oil in the back inlet. Move the pump forward in small increments until flow begins in the device and spherical microgels begin to form at the flow-focusing region. Start the pump with a 0.4 µL/min flowrate and let the device run until it stabilizes. If needed, adjust the flow rate ±0.1 µL/min in small increments to stabilize microgel production.
   7. Once microgel production stabilizes as shown in Figure 1B, replace the collection tube with a new tube, and turn on the UV light. Check the run periodically to ensure microgel production is stable over the duration of the run.

Figure 1: Microfluidic production of hyaluronic acid (HA) microgels with norbornene (NB) functional handles. (A) Approximately 31% of HA repeat units were successfully modified with NB, as determined by proton NMR analysis performed in deuterium oxide. ¹H NMR shifts of pendant norbornenes at δ6.33 and δ6.02 (vinyl protons, endo), and δ6.26 and δ6.23 ppm (vinyl protons, exo) were compared to the HA methyl group δ2.05 ppm to determine functionalization. Reprinted from Anderson et al.¹² with permission from Elsevier. (B) Schematic of the flow-focusing microfluidic device used to generate HA-NB µgels. (C) Maximum intensity projections from confocal microscopy were used to visualize fluorescently labeled µgels (scale bar = 500 µm). (D) Frequency distributions of microgel diameter from independent runs on the microfluidic setup demonstrate control over microgel size ~50 µm or ~100 µm depending on the device used. (E) Microgel diameter is reported as the mean and standard deviation for each independent run. Reprinted from Wilson et al.⁹ with permission from Wiley. Please click here to view a larger version of this figure.

3. Purifying and drying microgels

1. Purification of microgels
   1. Prepare the microgel washing buffer (300 mM HEPES, 50 mM NaCl, 50 mM CaCl₂) as well as 2% (w/v) Pluronic F-127 surfactant solution in washing buffer. Sterilize the solutions using a 0.2 µm vacuum-driven filter.
   2. Centrifuge the microgel collection tube (5,000 x g) for 5 min. In a sterile hood, carefully aspirate the supernatant oil phase. Combine the µgels 1:1 with 2% Pluronic F-127 surfactant solution and vortex to mix well. Centrifuge (5,000 x g) for 5 min and aspirate the supernatant washing solution.
   3. Add washing buffer at 4x microgel volume and vortex to mix well. Centrifuge (5,000 x g) the mixture for 5 min and aspirate the washing solution. Complete 4-8 washes with the washing buffer until the surfactant is removed from the system (i.e., no bubbles remain).
2. Fluorescent labeling of HA-NB microgels
   NOTE:The in-house synthesis of a fluorescently labeled tetrazine relies on two base-catalyzed thiol-Michael addition reactions in series that have been well described and previously reported³. For this work, Alexa Fluor-488 was conjugated with tetrazine for the labeling of norbornene-modified µgels. The lyophilized product (Alexa Flour 488-Tet) was dissolved in dimethylformamide at 1 mg/mL and stored at -20 °C.
   1. To fluorescently label the µgels, first prepare a working solution of Alexa Fluor 488-Tet by diluting the 1 mg/mL stock 1:14 in sterile 1x PBS. In a sterile hood, combine the µgels with the working solution (2:1 by volume).
   2. Use a displacement pipette and mix well. Incubate the mixture for 1 h at room temperature or overnight at 4 °C.
   3. Centrifuge (5,000 x g) and aspirate the staining solution. Wash the µgels twice with 1x PBS (1:1 by volume) to remove unreacted Alexa Fluor 488-Tet.
      NOTE: At this point, the fluorescently labeled µgels can be imaged on a confocal microscope to quantify the microgel size (Figure 1C-E)⁹. Methods for measuring microgel size have been thoroughly described by Roosa et al.¹⁷.
3. Drying HA-NB microgels
   1. Transfer purified µgels (Figure 2A) to a cryo-safe screw-cap tube using a positive displacement pipette. Add 70% ethanol to the purified µgels 50% (v/v) and mix well with a displacement pipette. Centrifuge for 5 min at 5,000 x g.
      CAUTION: Ethanol is a highly flammable substance.
      NOTE: The cryo-safe screw-cap tube can be weighed prior to adding µgels, and then weighed again after lyophilization to determine the mass of µgels. This is recommended to minimize error when using quantities less than 1 mg. Ensure that the scale is internally adjusted or calibrated prior to use.
   2. Aspirate the supernatant liquid and replace with 70% ethanol (50% v/v) (Figure 2B). Mix well with a displacement pipette. Incubate overnight at 4 °C.
      NOTE: Microgels can be stored in 70% ethanol at 4 °C prior to lyophilization for long-term storage, if needed. Lyophilized microgels are shown in Figure 2C. Other lyophilization media can be used in this step if cryogel formation is desired (Figure 2D).
   3. Briefly centrifuge to ensure the µgels are at the bottom of the screw-cap tube. Add liquid nitrogen to a cryogenic container, and then add the tube of µgels to flash-freeze.
   4. After 5-10 min, remove the tube of µgels with forceps. Quickly remove the cap and cover with a lab-grade tissue. Secure the tissue with a rubber band and transfer to a lyophilization container or chamber.
   5. Load the sample on the lyophilizer following the manufacturer's instructions. Lyophilize at 0.066 Torr and -63 °C. Store the lyophilized µgels (lyo-µgels) tightly sealed at room temperature.
      ​NOTE: Lyophilization is complete when all liquid is removed from the tube and a dried product remains. Organic solvents can decrease the longevity of the rubber fixtures on common lyophilization systems.

Figure 2: Drying HA-NB microgels. (A) Maximum intensity projection of µgels in aqueous solution (scale bar = 100 µm). (B) Purified µgels can be incubated 1:1 by volume in the lyophilization medium of choice and lyophilized. (C) Maximum intensity projection of dried lyo-µgels (scale bar = 100 µm). (D) Microgels are resuspended after lyophilization. EtOH (70%) is recommended for retaining the original properties of the µgels throughout the lyophilization process; however, other media such as isopropyl alcohol (IPA), water, and acetonitrile (MeCN) can be used interchangeably to facilitate cryogel formation (scale bar = 100 or 50 µm as noted). (E) Measurement of HA-NB microgel diameter before (gray) and after lyophilization (green) in 70% EtOH shown as frequency distributions for three microgel populations. Reprinted from Anderson et al.¹² with permission from Elsevier. Please click here to view a larger version of this figure.

4. MAP scaffold fabrication

1. Tetrazine linker synthesis
   NOTE: Tetrazine linkers can be used to interlink µgels bearing free norbornene groups (Figure 3A). HA-tetrazine (HA-Tet) synthesis procedure was adapted from Zhang et al.¹⁸ using 79 kDa sodium HA with molar equivalents of 1:1:0.25 of HA-repeat units to DMTMM to tetrazine-amine (Figure 3B)¹².
   1. Weigh the reactants. Dissolve the HA at 20 mg/mL in 200 mM MES buffer (pH ~6) by stirring in a beaker or flask on a stir plate. Once dissolved, add the DMTMM to the HA solution and allow to react for approximately 20 min at room temperature. For example, 100 mg HA + 72.8 mg DMTMM + 14.14 mg tetrazine-amine can be used.
   2. Dissolve the tetrazine-amine at 15 mg/mL in 200 mM MES buffer and add dropwise to the HA/DMTMM solution. Refer to Figure 3C for the HA-Tet reaction setup.
   3. Add parafilm to the opening of the reaction vessel to minimize evaporation and cover the reaction vessel with foil. Continue stirring while allowing the reaction to proceed for approximately 24 h.
   4. After 24 h, chill 200 proof ethanol (approximately 10x the reaction volume). On a stir plate, transfer the reaction dropwise to the chilled ethanol to precipitate the HA-Tet (Figure 3D) and continue stirring for 20 min.
   5. Transfer the solution to 50 mL conical tubes, and then centrifuge at 5,000 x g for 10 min. Pour off the excess ethanol to dispose as waste. Pull vacuum on the HA-Tet in a dessicator to dry overnight. An example of the dried product at this step in the protocol can be found in Figure 3E.
   6. Purify the HA-Tet using dialysis. Dissolve HA-Tet in 2 M NaCl solution and transfer to cellulose dialysis tubing with a 12-14 kDa molecular weight cut-off. Transfer the filled dialysis tubing to a bucket with 5 L of ultrapure water, and dialyze the HA-Tet against water overnight.
   7. The next day, remove the water and replace with 1 M NaCl solution for 30 min. Remove the NaCl solution, and then dialyze against ultrapure water for 3 days, replacing the water daily.
   8. Filter the dialyzed product using 0.2 µm vacuum-driven filter, and then transfer the filtered HA-Tet product to 50 mL conical tubes.
   9. Flash-freeze the conical tubes in liquid nitrogen for 10 min, and then remove the conical tubes with forceps. Quickly remove the cap and cover with a lab-grade tissue. Secure the tissue with a rubber band and transfer to a lyophilization container or chamber and lyophilize. Store the lyophilized product (Figure 3F) at -20 °C.
   10. Quantify tetrazine modification by dissolving the HA-Tet at 10 mg/mL in D₂O and analyzing via proton NMR (Figure 3G)¹⁶.
       1. To determine the amount of functionalization, first calibrate the D₂O solvent peak to 4.8 PPM. Integrate the peak for the HA methyl protons (δ2.05) and calibrate the integration to 3.0. Next, integrate the peaks for the pendant tetrazine groups at δ8.5 (2H) and δ7.7 (2H) (aromatic protons). Normalize the integration of these peaks to the corresponding number of protons to determine the average degree of modification¹².
2. Interlinking lyo-µgels to form MAP scaffolds for characterization
   1. Prepare the MAP scaffold components (i.e., µgels, HA-Tet, rehydration volume). Weigh the lyo-µgels (Figure 4A) and reconstitute in 84% of the final MAP volume of 1x PBS. Allow the microgels to swell for approximately 20 min (Figure 4B,C). The wt% MAP used for rehydration can be chosen based on the user's preference for final particle fraction (refer to Figure 4D, E).
   2. Dissolve the HA-Tet in 1x PBS at the chosen concentration (see NOTE below).
      NOTE: Changing both the packing fraction (via wt% MAP) as well as the concentration of HA-Tet will alter bulk scaffold mechanical properties. For example, a 3.4 wt% MAP scaffold crosslinked with 0.02 mg/mL HA-Tet (annealing ratio of 2.6 mol Tet:mol HA-NB) generates MAP scaffolds with approximately 700 Pa shear storage modulus¹².
   3. Use a displacement pipette to combine the HA-Tet and lyo-µgels and mix well. At this point, the mixture can be transferred via displacement pipette onto glass slides, well plates, or a container of the user's choosing. Allow µgels to anneal at 37 °C for 25 min, and then use a spatula to transfer the MAP scaffolds to well plates filled with 1x PBS. Keep MAP scaffolds in 1x PBS until ready for characterization.
3. Calculating MAP scaffold particle fraction
   1. For improved image quality, transfer MAP scaffold to a glass coverslip using a spatula. Image MAP scaffolds on a confocal microscope using the laser for FITC excitation and emission. Image MAP scaffolds on a 20x objective and obtain a Z-stack traversing 250-300 µm in the Z-direction with a step size of 2.5 µm. Make note of the µm/pixel calibration of the image.
   2. Import the Z-stack image into the analysis software (see Table of Materials). Select the Add New Surfaces button. Check the box to Segment Only a Region of Interest, and then select the blue arrow button Next: Region of Interest.
   3. Define a region of interest, keeping track of the X-, Y-, and Z-dimensions of the volume being analyzed. Select the blue arrow button Next: Source Channel.
      NOTE: X- and Y-dimensions are in units of pixels while the Z-dimension is the number of steps. A recommended Z-height for the region of interest should include a minimum of two µgels.
   4. Use the Source Channel drop-down list to select the FITC channel. Check the box next to Smooth and input a surface detail of 2.50 µm. Under Thresholding, select Absolute Intensity, and then select the blue arrow button Next: Threshold.
   5. Use the suggested thresholding value for the FITC channel. Rotate the 3D projection to assess the rendering quality and adjust as needed. Select Next: Classify Surfaces.
      NOTE: The Back button can be used to edit previous steps in the process, such as Z-dimension, as needed.
   6. Check whether Number of Voxels is 10.0, and then select the green double arrow button Finish: Execute all creation steps and terminate the wizard.
      NOTE: Volume rendering parameters can be stored for batch analysis so that the same settings are applied to analyze all the scaffolds.
   7. To export the data, select the Statistics tab, and then the Detailed tab. Use the second drop-down box to select the variable Volume. Select the floppy disk button Export Statistics on Tab Display to File and save as a spreadsheet file (.xls) when prompted.
   8. Open the file and use the SUM function on Column A Volume to determine the total volume (µm³) of the µgels in the region of interest.
   9. Convert the dimensions of the region of interest that was analyzed from pixels to µm. Use the µm/pixel calibration of the image from step 4.3.1 to convert the X- and Y-dimensions. Multiply the Z-dimension (number of steps) by the step size for the image to convert the Z-dimension to µm. Calculate the volume of the region of interest (µm³) by multiplying the X-, Y-, and Z-dimensions.
   10. To determine the particle fraction of the scaffold, divide the total volume of the µgels in the region of interest (found in step 4.3.8) by the volume of the region of interest (found in step 4.3.9).

Figure 3: Synthesis of tetrazine linker for the fabrication of microporous annealed particle (MAP) scaffolds. (A) Schematic of HA-NB µgels being interlinked with a tetrazine linker to form MAP scaffolds. (B) Reaction scheme for HA-Tet synthesis. (C) The HA-Tet reaction was setup and allowed to react overnight followed by (D) precipitation of HA-Tet in ethanol. (E) Once purified and dried, the HA-Tet was rehydrated and lyophilized to yield (F) a dried, light pink product. (G) Proton NMR analysis shows successful modification of 11% of HA repeat units. Reprinted from Anderson et al.¹² with permission from Elsevier. Please click here to view a larger version of this figure.

Figure 4: Rehydration of lyophilized microgels for MAP scaffold fabrication. (A) Maximum intensity projection of dried lyo-µgels (scale bar = 100 µm). (B) After freeze-drying, rehydration of lyo-µgels is shown to take approximately 20 min (scale bar = 100 µm). (C) Lyo-µgels can be rehydrated at varying wt% MAP to produce jammed µgels (scale bar = 100 µm). (D) Increasing the wt% MAP when rehydrating lyo-µgels alters the particle fraction in MAP scaffolds, as shown by single Z-slices of MAP scaffolds and volume projections (scale bar = 100 µm). (E) Using these user-defined wt% MAP scaffolds, unique particle fractions can be achieved (NL = non-lyophilized µgels). A one-way ANOVA with Tukey HSD was performed on the samples (n = 3), with significance reported at p < 0.05 (*), p < 0.01 (**), p < 0.005 (***), and p < 0.001 (****). Reprinted from Anderson et al.¹² with permission from Elsevier. Please click here to view a larger version of this figure.

5. 3D cell culture in map scaffolds

1. Prepare cell culture devices
   1. To create a custom cell culture device for these experiments (Figure 5A-C), use a 3D printer to print a negative mold using the CAD file found in Supplemental Coding File 1.
      NOTE: The dimensions of the cell culture device are as follows: 94.9 mm x 94.9 mm x 4.8 mm with 2.6 mm total well height. The diameter of the inner wells and outer wells are 4 mm and 6 mm, respectively.
   2. Mix polydimethylsiloxane (PDMS) elastomer base with the curing agent at a 10:1 ratio by mass. Pour the PDMS mixture into a large plastic Petri dish and degas in a desiccator for approximately 30 min or until all the bubbles have disappeared.
   3. Once all the bubbles have disappeared, carefully place the 3D printed mold into the PDMS to minimize the formation of new bubbles. Place in the oven at 60 °C for at least 2 h to cure the PDMS.
   4. Use a knife or razor blade to gently trace around the parameter of the culture device, and then carefully remove the mold. Use a 4 mm biopsy punch to remove any PDMS from the bottom of the wells. Cut the devices to fit on a glass coverslip.
      NOTE: Cell culture devices can also be bonded to glass slides, but glass coverslips improve sample imaging.
   5. Use tape to remove dust from the bottom side of the culture devices. Place the clean glass coverslips and culture devices (bottom side up) on a hot plate at 135 °C for at least 15 min to remove moisture.
   6. In a fume hood, use a corona plasma gun on high on both the glass coverslip and the bottom side of the device for 30 s, and then quickly bond the treated surfaces together. Gently apply pressure to ensure a good seal between the culture device and glass coverslip.
   7. Repeat step 5.1.6 for all devices, and then place in a 60 °C oven overnight to secure the bond. Autoclave the devices to sterilize before use in vitro.
2. Cell culture in MAP scaffolds
   1. Prepare the MAP scaffold components (i.e., µgels, HA-Tet, media volume) based on the desired particle fraction (refer to Figure 4D-E). Weigh the lyo-µgels in a sterile hood and reconstitute in 84% of the final MAP volume of cell media based on the chosen wt% MAP. Allow the µgels to swell for approximately 20 min.
      NOTE: These methods require the user to weigh the lyo-microgel product for rehydration. For small masses (1 mg or less), it is suggested to first weigh the cryotube before adding and lyophilizing µgels, and then reweigh the tube after lyophilization to determine the mass of the product to minimize error.
   2. Dissolve the HA-Tet in cell media in 16% of the final MAP volume.
      NOTE: The following steps for preparing cells for seeding in MAP scaffolds can be altered depending on the cell type being used. In this protocol, D1 mouse mesenchymal cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% penicillin-streptomycin (pen-strep) and 10% fetal bovine serum (FBS) (see Table of Materials). Standard adherent cell culture protocols should be followed for these cells, with the cultures maintained at 37 °C and 5% CO₂ in tissue culture-treated culture vessels.
   3. Once D1 mouse mesenchymal cells have reached 70%-80% confluency, aspirate the media and wash the cells with 1x PBS. Lift the cells by adding enough volume of 1% trypsin-EDTA to cover to surface of the tissue culture vessel. Incubate at 37 °C for 1-3 min, and then quench the trypsinization by adding DMEM media supplemented with 1% pen-strep and 10% FBS at 2x the volume of trypsin-EDTA.
   4. Centrifuge the cell suspension at 100 x g for 5 min at room temperature to pellet the cells. Aspirate the supernatant media and resuspend the cells in 1 mL DMEM media supplemented with 1% pen-strep and 10% FBS.
   5. Ensure the cell suspension is well mixed, and then transfer 20 µL to a new microcentrifuge tube. Add 20 µL trypan blue solution and mix well. Use 20 µL of this mixture to count the cells using either a hemocytometer or an automated cell counter with cell counting chamber slides.
   6. Transfer the number of cells needed for seeding 10,000 cells/µL MAP to a new microcentrifuge tube. Centrifuge at 100 x g for 5 min at room temperature to pellet the cells. Carefully aspirate the supernatant media from the cell pellet without aspirating the cells.
   7. Add the µgels and crosslinker to the cell pellet with a displacement pipette. Mix well with a displacement pipette, and then seed 10 µL of the mixture per well. When plating, pipette in a circular motion to evenly distribute the mixture in the well.
   8. Allow the µgels to anneal at 37 °C in the cell incubator for 25 min before adding cell media to fill the wells (~50 µL of media per well). Maintain the 3D cultures at 37 °C and change media as needed. To avoid aspirating the scaffold when changing media, stabilize the pipette tip along the ridge of the upper well.
      NOTE: When adding or removing liquid from the culture wells, rest the end of the pipette tip on the ledge above the MAP scaffold to minimize the chance of disrupting or aspirating the scaffold from the well.
   9. At the desired time points, fix samples by removing the media and adding 50 µL of 4% paraformaldehyde per well for 30 min at room temperature. Wash the samples 3x with 50 µL of 1x PBS or preferred buffer. At this point in the protocol, standard methods for immunofluorescence or fluorescence staining can be followed, using 50 µL per well as the working volume.
      NOTE: These methods for fixation and cell staining specifically describe the use of fluorescent stains; however, immunostaining with primary and/or secondary antibody conjugations can be performed in these scaffolds as well following the manufacturer's instructions using 50 µL as the working volume per well.
   10. Image cells in MAP scaffolds on a confocal microscope using a 20x objective and obtain a Z-stack traversing 200-250 µm in the Z-direction with a step size of 2.5 µm. An example of fluorescence staining with DAPI (nuclear stain diluted 1:1000 in 0.15% Triton-X in 1x PBS) and phalloidin-647 (F-actin stain diluted 1:40 in 0.15% Triton-X in 1x PBS) is shown in Figure 5E, F with fixed D1 cells cultured in MAP scaffolds for 3 days.
       NOTE: Plasma treatment of glass surfaces results in increased hydrophilicity, which has been shown to enhance cell adhesion. Cells will likely be observed spreading along the bottom of the cell culture wells but should not be included in cell counts or cell volume quantification for assessing cell response in MAP scaffolds.

Figure 5: Cell culture in MAP scaffolds. (A) The mold for creating cell culture wells can be 3D printed and cast with PDMS. The entire mold is 95 mm in diameter, the large wells are 6 mm in diameter, and the small inner wells are 4 mm in diameter. (B) Once cast with PDMS, the cell culture devices are plasma bonded to coverslips for improved microscopy capabilities. (C) The cross section of a cell culture well depicts the reservoir for cell media (~50 µL) and a smaller reservoir for seeding MAP scaffold with cells (~10 µL). (D) The process of seeding cells in MAP scaffolds first relies on the rehydration of lyo-µgels at the user's desired wt%, followed by mixing with cells and the crosslinker for interlinking the µgels. (E) Cells can be encapsulated in MAP scaffolds (green) with varied wt% MAP. Representative images are from day 5 of D1 cell culture in MAP scaffolds (scale bar = 100 µm). (F) Single Z-slices show differences in cell growth in scaffolds comprising different wt% MAP (scale bar = 50 µm). Reprinted from Anderson et al.¹² with permission from Elsevier. Please click here to view a larger version of this figure.

Representative Results

The aim of this protocol is to demonstrate the preparation of microporous annealed particle (MAP) scaffolds with a bio-orthogonal crosslinking scheme as well as controlled particle fractions for 3D cell culture. First, HA was modified with norbornene pendant groups to be used in both microgel formation and interlinking to form MAP scaffolds. Using these methods, approximately 31% of HA repeat units were successfully modified with a norbornene functional handle (Figure 1A). Microfluidic devices with a flow-focusing region (Figure 1B) were shown to produce HA-NB µgels of either ~50 µm or ~100 µm in diameter (Figure 1C,D). The µgels used throughout the rest of this work had a mean diameter of 92 µm (Q1 = 79 µm, Q3 = 103 µm) (Figure 1E).

To control the particle fraction, µgels were dried via lyophilization (Figure 2A-C) to produce a product that could be weighed by the user and rehydrated to achieve swollen µgels. The medium for lyophilizing the µgels was optimized to prevent cryogel formation (i.e., internal defects) by using 70% ethanol, but it has also been demonstrated that cryogels were achieved using other media for lyophilization if cryogels are desired by the user (Figure 2D). A comprehensive study of different lyophilization media for microgel cryogel formation can be found in the work by Anderson et al.¹². Using confocal microscopy, HA-NB microgel diameter was quantified both before and after lyophilization with 70% ethanol (Figure 2E), which indicated no significant change in microgel size with this drying process.

To facilitate bio-orthogonal interlinking of HA-NB µgels, a linear HA-Tet crosslinker was synthesized (Figure 3). Proton NMR spectroscopy showed successful modification of 11% of HA repeat units with a tetrazine pendant group using the steps detailed in this work (Figure 3G), and the reaction yield was 95%. Using the HA-Tet linker, the dried lyo-microgel product (Figure 4A) was rehydrated with specified volumes to achieve different wt% (w/v) formulations of µgels (Figure 4B,C) in MAP scaffolds ranging from 10 µL (4 mm diameter) to 50 µL (8 mm diameter) in size for cell culture experiments and material characterization, respectively. These user-defined wt% of µgels in MAP scaffolds corresponded to unique particle fractions in the scaffolds (Figure 4D,E).

This protocol also detailed the process for seeding cells in MAP scaffolds for 3D culture (Figure 5D) using bio-orthogonal annealing schemes. The lyo-µgels were rehydrated at the desired wt% in the cell media, and then mixed with the cell pellet and HA-Tet for interlinking the µgels. This mixture was then plated in cell culture devices (Figure 5A-C) to yield cells encapsulated in the void space between µgels in MAP scaffolds. Cells in 3D culture in MAP scaffolds were fixed on day 5, stained, and imaged on a confocal microscope as shown in Figure 5E. An example of the cells in the void space of the MAP scaffolds for different wt% formulations is shown in Figure 5F.

Supplemental Coding File 1: CAD file used to 3D print the mold for cell culture devices. Please click here to download this File.

Discussion

Microfluidic production of HA-NB microgels has been shown to generate microgels with a narrower range of size distribution than emulsion batch production^3,9. The microgels described in this protocol were formulated using an MMP-cleavable crosslinker (Ac-GCRDGPQGIWGQDRCG-NH₂) to support material degradation. However, HA-NB microgels can also be crosslinked using an alternative di-thiol linker such as dithiothreitol (DTT), which is non-degradable. Similarly, other photo-initiators, such as Irgacure 2959 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, can be used in the HA-NB precursor instead of LAP to facilitate thiol-ene crosslinking¹⁰. These microgels were composed of 3.5 wt% HA based on a previous work that demonstrated the viscosity of a 3.5 wt% precursor solution was amenable for pinching into droplets on a microfluidic platform⁶. Others have described HA-NB microgel fabrication on flow-focusing microfluidic devices using lower wt% formulations (3 wt% HA-NB) as well^8,10,11. While this method is beneficial for producing microgel populations with low polydispersity, it is more time- and labor-intensive than batch emulsion techniques that can be used for HA-NB microgel production as well³.

The microgel lyophilization process relies on freezing crosslinked polymer networks. Defects can occur during freezing when crystal structures in the lyophilization medium form and act as porogens, and the resulting cryogels exhibit enhanced porosity and different mechanical properties compared to the original material^19,20. To date, there have been methods described for freeze-drying microgels that prevent cryogel formation for polyethylene glycol (PEG)¹³, GelMa¹⁴, and HA formulations¹² using different mediums for lyophilization. For this protocol, the user could interchange 70% ethanol with another lyophilization medium of their choosing if cryogels are desired. While cryogels can be advantageous for some applications, 70% ethanol was highlighted in this protocol as the lyophilization medium because it is a common lab reagent, it incorporates the benefit of sterilization in the process of MAP fabrication, and it allows the HA microgels to retain their original size, shape, and stiffness while also minimizing internal defects once rehydrated¹². In addition to these assessments of microgel characteristics, environmental scanning electron microscopy (SEM) could be implemented as a potential tool to assess microgel morphology before and after lyophilization.

The methods described in this work for drying HA-NB microgels were introduced to circumvent the variability in particle fraction and produce consistent MAP scaffolds with user-defined wt% formulations. The study of controlled particle fraction at different wt% rehydration was limited to the study of spherical microgels. Other microgel shapes, such as rods^8,21 or irregular shapes^10,22, have not been investigated to assess the relationship between wt% MAP and particle fraction. Particle fraction has been assessed in MAP scaffolds using consistent proportions of rehydrated lyo-microgels (84%) and HA-Tet crosslinker (16%) based on a previous study²; however, these fractions could be altered at the user's discretion as long as the HA-Tet volume is sufficient for dissolving HA-Tet at the desired crosslinking ratio. The norbornene-tetrazine click chemistry reaction has been effective in annealing microgels to form MAP scaffolds using bi-functional²³, multi-arm³, as well as the linear¹² tetrazine linker described in these methods. If desired, the molar ratio of Tet:HA-NB can be varied to achieve different stiffnesses of the bulk MAP scaffolds¹². These techniques for rehydrated lyo-microgels have yet to be assessed for other annealing chemistries besides tetrazine-norbornene.

3D cell culture in MAP scaffolds has been described previously with numerous cell types, such as endothelial cells⁸, fibroblasts^1,3,4,24, neural progenitor cells⁹, and mesenchymal stem cells^25,26. Residual ethanol was observed via proton NMR after HA-NB microgels were lyophilized in 70% ethanol; however, it was also confirmed that the trace amounts of ethanol did not impact cell viability¹². Culture in MAP scaffolds comprising lyo-microgels has only been demonstrated thus far with mouse mesenchymal stem cells¹²; however, this protocol for seeding cells in MAP scaffolds with lyo-microgels could be interchanged with other cell types and their corresponding cell media. For D1 cell culture, F-actin intensity and total cell volume has been shown to decrease as the wt% MAP increases¹². It has also been shown that increasing the wt% MAP corresponds to an increase in bulk scaffold stiffness (not local microgel stiffness) as the void space between microgels becomes smaller, leading to less cell proliferation in high wt% MAP scaffolds¹².

This work described methods for producing HA-NB microgels on a microfluidic platform followed by freeze-drying the microgels to either retain the original microgel properties or produce cryogels. This protocol outlined the synthesis of a tetrazine linker used to interlink the lyo-microgels once they have been rehydrated at user-defined weight percentages to create MAP scaffolds with controlled particle fractions. Lastly, this work detailed the steps for culturing cells within these MAP scaffolds with user-defined microarchitectures. Creating granular scaffolds with consistent particle fractions improves the reproducibility of experimental results for MAP users, extending beyond cell responses to mass transport and mechanical properties as well¹². MAP scaffolds generated using these techniques can be used moving forward for in vivo applications. The use of a lyophilized product for granular scaffold fabrication is beneficial for improving the material shelf-life and will also be advantageous for future translation of MAP scaffolds to a clinical setting.

Materials

Name	Company	Catalog Number	Comments
1 mL Luer-Lok syringe sterile, single use, polycarbonate	BD	309628
5 mL Luer-Lok syringe sterile, single use, polycarbonate	BD	309646
Alexa Fluor 488 C5 maleimide	Invitrogen	A10254	For synthesis of fluorescently-labeled tetrazine
Alexa Fluor 647 Phalloidin	Invitrogen	A22287	For staining cell culture samples
Aluminum foil	VWR	89107-726
Biopsy punch with plunger, 1.0 mm	Integra Miltex	69031-01
Biopsy punch, 4 mm	Integra Miltex	33-34
Blunt needle, 23 G 0.5", Non-Sterile, Capped	SAI Infusion Technologies	B23-50
Bottle-top vacuum filter, 0.22 μm	Corning	CLS430521
Calcium chloride	VWR	1B1110	For microgel washing buffer
Capillary-piston assemblies for positive-displacement pipettes, 1000 μL max. volume	Rainin	17008609
Capillary-piston assemblies for positive-displacement pipettes, 25 μL max. volume	Rainin	17008605
Capillary-piston assemblies for positive-displacement pipettes, 250 μL max. volume	Rainin	17008608
Countess Cell Counting Chamber Slides	Invitrogen	C10228
Countess II FL Automated Cell Counter	Invitrogen	AMQAF1000
Centrifuge tube, 15 mL	CELLTREAT	667015B
Centrifuge tube, 50 mL	CELLTREAT	229421
Chloroform, ACS grade, Glass Bottle	Stellar Scientific	CP-C7304	For synthesis of fluorescently-labeled tetrazine
Corona plasma gun, BD-10A High Frequency Generator	ETP	11011
CryoTube Vials, Polypropylene, Internal Thread with Screw Cap	Nunc	368632
D1 mouse mesenchymal cells	ATCC	CRL-12424	Example cell line for culture in MAP gels
DAPI	Sigma-Aldrich	D9542	For staining cell culture samples
Deuterium oxide, 99.9 atom% D	Sigma-Aldrich	151882	For NMR spectroscopy
Dialysis tubing, regenerated cellulose membrane, 12-14 kDa molecular weight cut-off	Spectra/Por	132703	For purifying HA-NB and HA-Tet
Diethyl ether	VWR	BDH1121-4LPC	For synthesis of fluorescently-labeled tetrazine
Dimethylformamide	Sigma-Aldrich	277056	For synthesis of fluorescently-labeled tetrazine
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)	TCI-Chemicals	D2919	For modifying HA
Dithiothreitol (DTT)	Thermo Scientific	R0861	Non-degradable dithiol linker (substitute for MMP-cleavable peptide)
Dulbecco's Modified Eagle's Medium (DMEM), high glucose, w/ 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture	Sigma-Aldrich	D6429-500ML	For D1 cell culture
EMS Paraformaldehyde, Granular	VWR	100504-162	For making 4% PFA
Ethanol absolute (200 proof)	KOPTEC	89234-850
Fetal bovine serum (FBS)	ATCC	30-2020	For D1 cell culture
Heating Plate	Kopf Instruments	HP-4M
Hemacytometer with coverglass	Daigger Scientific	EF16034F
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)	Sigma-Aldrich	H3375
Sodium hyaluronate, 79 kDa average molecular weight, produced in bacteria Streptococcus zooepidemicus, pharmaceutical grade, microbial contamination <100 CFU/g, bacterial endotoxins <0.050 IU/mg	Contipro	N/A	79 kDa average molecular weight was used for HA-Tet synthesis, but these methods could be adapted for other molecular weights.
IMARIS Essentials software package	Oxford Instruments	N/A	Microscopy image analysis software
Infusion pump, dual syringe	Chemyx	N/A
Kimwipe	Kimberly-Clark	34120
Laboratory stand with support lab clamp	Geyer	212100
Liquid nitrogen	Airgas	NI 180LT22
Lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate	TCI-Chemicals	L0290
Lyophilizer	Labconco	N/A	Labconco FreeZone 6 plus has been discontinued, but other lab grade console freeze dryers could be used for this protocol.
Methyltetrazine-PEG4-maleimide	Kerafast	FCC210	For synthesis of fluorescently-labeled tetrazine
2-(4-Morpholino)ethane Sulfonic Acid (MES)	Fisher Scientific	BP300-100	For modifying HA
Micro cover glass, 24 x 60 mm No. 1	VWR	48393-106
Microfluidic device SU8 master wafer	FlowJem		Custom design made either in-house in clean room or outsourced
Mineral oil, heavy	Sigma-Aldrich	330760
MMP-cleavable dithiol crosslinker peptide (Ac-GCRDGPQGIWGQDRCG-NH2)	GenScript	N/A
5-Norbornene-2-methylamine	TCI-Chemicals	95-10-3	For HA-NB synthesis
Packing tape	Scotch	3M 1426
Parafilm	Bemis	PM996
PEG(thiol)2	JenKem Technology USA	A4001-1	For synthesis of fluorescently-labeled tetrazine
Penicillin-Streptomycin, 10,000 units/mL	Thermo Fisher Scientific	15140122	For D1 cell culture
Petri dish, polystyrene, disposable, Dia. x H=150 x 15 mm	Corning	351058
Pluronic F-127	Sigma-Aldrich	P2443	For washing HMPs
Phosphate buffered saline (PBS) 1x	Gibco	10010023
RainX water repellent glass treatment	Grainger	465D20	Synthetic hydrophobic treatment solution for microfluidic device treatment
RGD peptide (Ac-RGDSPGERCG-NH2)	GenScript	N/A
Rubber bands	Staples	112417
Sodium chloride	Chem-Impex	30070	For dialysis
Span 80 for synthesis	Sigma-Aldrich	1338-43-8
Sylgard 184 Silicone Elastomer	Electron Microscopy Science	4019862	polydimethylsiloxane (PDMS) elastomer for making microfluidic devices and tissue culture devices
Syringe filter, Whatman Uniflo, 0.2 μm PES, 13 mm diameter	Cytvia	09-928-066
Tetraview LCD digital microscope	Celestron	44347
Tetrazine-amine HCl salt	Chem-Impex	35098	For HA-Tet synthesis
Triethylamine	Sigma-Aldrich	471283	For synthesis of fluorescently-labeled tetrazine
Tris(2-carboxyethyl)phosphine (TCEP)	Millipore Sigma	51805-45-9
Triton X-100	VWR	97063-864
Trypan blue solution, 0.4%	Thermo Fisher Scientific	15250061
Trypsin EDTA (0.25%), Phenol red	Fisher Scientific	25-200-056	For lifting adherent cells to seed in MAP gels
Tygon ND-100-80 Non-DEHP Medical Tubing, Needle Gauge=23, Wall Thickness=0.020 in, Internal diameter = 0.020, Outer diameter = 0.060 in	Thomas Scientific	1204G82
UV curing system controller, LX500 LED	OmniCure	010-00369R
UV curing head, LED spot UV	OmniCure	N/A
UV light meter, Traceable	VWR	61161-386
Vacuum dessicator	Bel-Art	08-594-15C
X-Acto Z Series Precision Utility Knife	Elmer's	XZ3601W