Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

1. Decellularized Tissue Processing

1. Decellularization and Lyophilization
   1. Decellularize tissue(s) of interest following an established protocol.
      NOTE: Scaffolds in the current study have been prepared following published decellularization protocols for human DAT²⁸, porcine DDT⁸, and porcine DLV³². Commercially-available, insoluble collagen can also be used to fabricate foams and microcarriers, such as collagen sourced from bovine tendon, which has been used successfully over a concentration range of 10 – 50 mg/mL. Other insoluble collagen sources may be utilized, but may require optimization to identify the concentration range that will yield stable scaffolds.
   2. Transfer the hydrated decellularized tissue or purified collagen into a 50 mL centrifuge tube with forceps and add sufficient double distilled water (ddH₂O) to submerge the tissue, to a maximum total volume of 35 mL.
   3. Freeze the sample overnight at -80 °C, with the centrifuge tube positioned horizontally during freezing to maximize the surface area for sublimation during subsequent lyophilization.
   4. Remove the cap from the centrifuge tube and transfer the frozen sample into a lyophilization jar.
   5. Lyophilize the sample using a laboratory freeze dryer for 48 – 72 h or until fully dried.
      NOTE: Drying time may vary for different lyophilizers and decellularized tissues.
   6. Finely mince the lyophilized ECM into small pieces (~ 1 – 2 mm³) using sharp surgical scissors.
   7. Store the minced ECM in a desiccator until ready for further processing.
      NOTE: It is recommended that the minced ECM be used immediately to prevent moisture absorption from the environment.
2. Cryomilling (optional for foam fabrication)
   1. Fill a 25 mL stainless steel milling chamber for a laboratory ball mill system with minced lyophilized ECM and add two 10 mm stainless steel milling balls.
   2. Close and completely submerge the loaded milling chamber in liquid nitrogen for 3 min.
   3. Mill the frozen sample for 3 min at 30 Hz (1,800 rpm).
   4. Repeat steps 1.2.2 and 1.2.3 until the ECM is milled into a fine powder.
      NOTE: The number of times these steps should be repeated may vary between ECM sources. For example, DAT typically requires 3 milling cycles to generate a fine powder.
   5. Transfer the powder using a scoopula into a glass vial, seal tightly, and store in a desiccator.
3. ECM Suspension Preparation
   1. Weigh out 250 mg of either minced (for foams) or cryomilled (for foams or microcarriers) ECM and transfer it to a 15 mL centrifuge tube. Refer to step 1.1.6 and section 1.2 for preparation of minced ECM and cryomilled ECM, respectively.
      NOTE: This protocol is designed to prepare a 50 mg/mL ECM suspension, which is recommended for human DAT, porcine DDT, and porcine DLV. Depending on the specific ECM source, uniform suspensions may be generated at higher or lower starting concentrations.
   2. Add 5 mL of 0.22 M NaH₂PO₄ (pH 5.4) buffer to the centrifuge tube and mark the liquid level on the tube.
   3. Prepare an α-amylase stock solution by adding 7.5 mg of α-amylase to 1 mL 0.22 M NaH₂PO₄ (pH 5.4) buffer. Add 100 μL of the α-amylase stock solution (0.75 mg of α-amylase; 0.3% w/w of dry tissue) to the sample. Add 0.22 M NaH₂PO₄ to obtain a final volume of 10 mL.
   4. Agitate the suspension continuously at 300 rpm for 72 h at room temperature.
   5. Centrifuge the suspension at 1,500 x g for 10 min. Carefully collect and discard the supernatant without disturbing the digested ECM pellet. Resuspend the pelleted material in 10 mL of 5% NaCl diluted in ddH₂O.
   6. Repeat step 1.3.5 for a total of two rinses with 5% NaCl in ddH₂O.
   7. Discard the supernatant and resuspend the pelleted material in 10 mL of ddH₂O.
   8. Agitate the suspension continuously at 300 rpm for 10 min at RT.
   9. Centrifuge the suspension at 1,500 x g for 10 min. Carefully collect and discard the supernatant without disturbing the digested ECM pellet.
   10. Add 0.2 M acetic acid to the 5 mL mark made in step 1.3.2.
   11. Agitate the suspension continuously at 120 rpm O/N at 37 °C.
   12. Homogenize the ECM suspension at room temperature in ten-second intervals using a hand-held homogenizer equipped with a saw tooth 10 mm wide probe until no visible fragments remain. Place the suspension in a beaker of cold water between the homogenization intervals to prevent overheating.
       NOTE: Depending on the ECM source, further dilution in 0.2 M acetic acid may be required to obtain a homogeneous suspension. Excessive cooling of the ECM suspension may result in an increase in viscosity that can interfere with efficient homogenization. If an increase in viscosity is noted, the sample can be rewarmed to 37 °C and subjected to further processing.
   13. Store at 4 °C for up to one month prior to foam or microcarrier fabrication.

2. ECM-derived Foam Fabrication

1. Incubate the ECM suspension from step 1.3.13 (minced or cryomilled) at 37 °C with continuous agitation at 120 rpm until the suspension is warm.
2. Dilute the ECM suspension in 0.2 M acetic acidic to the desired concentration.
   NOTE: Depending on the ECM source, stable foams may be prepared in the range of 10 – 100 mg/mL, with 15 – 50 mg/mL as the recommended range for DAT, DDT and DLV. Typically, foams fabricated at higher concentrations will be slightly less porous but more stable in culture and easier to handle.
3. Using a 3 mL syringe with an 18 G needle to prevent bubble formation in the ECM suspension, fill the desired mold with the ECM suspension. To fabricate the DAT, DDT, and DLV foams, dispense 400 μL of 35 mg/mL ECM suspension into a 48-well cell culture-treated plate.
   NOTE: The shape of the selected mold and the volume of ECM suspension will determine the geometry of the resultant foam.
4. Cover and freeze the molds overnight by placing them in a -20 °C or -80 °C freezer.
   NOTE: The freezing temperature may affect the porosity of the foams. Larger pores are expected when the samples are frozen at -20 °C as compared to -80 °C due to the formation of larger ice crystals³³. To fabricate foams with a uniform porous structure, ensure that the mold is not in contact with a conductive surface to prevent directional cooling.
5. Place the molds containing the frozen samples into a lyophilizer flask. Connect the lyophilizer flask to the laboratory freeze dryer system and dry for 24 h.
   NOTE: It is important that the sample remains frozen prior to lyophilization.
6. Store the lyophilized foams in a desiccator until required.

3. ECM-derived Microcarrier Fabrication via Electrospraying

NOTE: An overview of the electrospraying set up is shown in Figure 2.

1. Incubate the cryomilled ECM suspension from step 1.3.13 at 37 °C with continuous agitation at 100 rpm O/N.
   NOTE: Cryomilled ECM is used to fabricate the ECM-derived microcarriers to ensure greater uniformity in the suspension and prevent clogging during electrospraying.
2. Load 3 mL of ECM suspension into a 3 mL Luer lock syringe and attach a winged infusion set onto the bore of the syringe.
   NOTE: A range of needle gauges can be used, recognizing that the selection may influence the size and shape of the resultant microcarriers. To fabricate the DAT, DDT, and DLV microcarriers, use a 25 G needle.
3. Secure the syringe within the syringe pump. Fasten the needle to a retort stand and position the needle tip vertically at a distance of 4 – 6 cm from the top of a low-form Dewar flask (250 – 500 mL size range recommended).
4. Attach an alligator clip electrode to the tip of the needle and connect it to the positive terminal of the high-voltage power supply.
5. Fold a strip of aluminum foil (12 x 5 cm) over the edge of the vacuum flask.
6. Attach a second alligator clip electrode to the outer edge of the foil and connect it to the ground source terminal of the power supply.
7. Fill the Dewar flask with liquid nitrogen to approximately 1 cm from the top, so that ¾ of the foil is submerged.
   NOTE: It is important to keep the Dewar flask filled with liquid nitrogen. The surface of the liquid nitrogen must be no less than 2 cm from the top of the flask during the electrospraying process. Continuous refilling of the liquid nitrogen is required to maintain a constant level during electrospraying.
8. Set the syringe pump to an infusion rate of 30 mL/h.
   NOTE: Varying the infusion rate can alter the microcarrier shape and diameter, but the recommended range is 25 – 35 mL/h. While it is highly dependent on the viscosity of the suspension, flow rates below 25 mL/h may result in the generation of larger microcarriers. At very low flow rates, the size and shape of the microcarriers may become less homogeneous. At infusion rates above 35 mL/h, the uniformity of the droplets generated via electrospraying may be disrupted, resulting in non-spherical microcarriers with a broader size distribution³⁴.
9. Apply a voltage of 20 kV and turn on the syringe pump to initiate electrospraying.
   NOTE: The voltage may be varied to tune the size of the microcarriers and tends to have a greater impact on the diameter than the infusion rate. The recommended voltage range is 15 – 25 kV. A decrease in microcarrier diameter is expected with an increase in voltage³⁵.
10. Once the infusion is complete, carefully pour off excess liquid nitrogen from the Dewar, leaving the microcarriers suspended in ~ 25 mL of liquid nitrogen to ensure they remain frozen.
11. Immediately transfer the microcarriers with the liquid nitrogen into a 50 mL centrifuge tube by pouring in one smooth motion. Collect any frozen microcarriers remaining in the Dewar with a scoopula and add them to the centrifuge tube.
    NOTE: Depending on the size of the Dewar, the microcarriers in liquid nitrogen can be initially poured into a mid-sized vessel, and then transferred immediately into the 50 mL centrifuge tube.
12. Cover the centrifuge tube containing the microcarriers in liquid nitrogen with aluminum foil perforated with small holes in preparation for lyophilization.
13. Place the covered centrifuge tubes into a lyophilizer flask. Immediately connect the flask to the lyophilizer and dry the samples O/N.
    NOTE: It is very important to keep the microcarriers suspended in liquid nitrogen to ensure they do not thaw before this step, as thawing may result in collapse and/or aggregation. DAT, DDT and DLV microcarriers fabricated at a concentration of 35 mg/mL using the electrospraying conditions described above will typically have a hydrated diameter ranging between 350 – 500 μm in size. The size distribution may vary depending on the ECM source.
14. Store the lyophilized microcarriers in a desiccator until required.

Figure 2. Overview of the Electrospraying Apparatus used in Microcarrier Fabrication. A: Image showing the arrangement of the key electrospraying equipment including the syringe pump and high voltage power supply, as well as the positioning of the needle relative to the Dewar of liquid nitrogen. B: Electrospraying schematic, including the recommended ranges for the voltage, infusion rate, and distance. Please click here to view a larger version of this figure.

4. Preparing Foams and Microcarriers for Cell Culture

1. Rehydration
   1. Transfer the lyophilized foams with forceps into a 50 mL centrifuge tube containing excess absolute ethanol (~ 99.9%) (~ 5:1 ratio of ethanol to foams). Similarly, resuspend the lyophilized microcarriers in excess absolute ethanol within the original centrifuge tube used for collection. Using a serological pipette, filter the microcarriers through a stainless sieve with a defined mesh size into a new 50 mL centrifuge tube to remove any aggregates and select for desired size ranges.
      NOTE: If the foams are fabricated within TCPS plates, they can be rehydrated directly within these vessels.
   2. Incubate at 4 °C for 4 h or until the foams or microcarriers have gravity settled to the bottom of the centrifuge tube. Perform all subsequent steps in a laminar flow hood using sterile reagents and proper aseptic technique.
   3. Remove the absolute ethanol using a serological pipette and add excess (5:1 ratio) 95% ethanol diluted with sterile phosphate buffered saline (PBS) to the scaffolds. Incubate at 4 °C for 4 h or until the ECM-derived scaffolds have sunk to the bottom of the rehydration vessel.
   4. Gradually rehydrate the scaffolds through an ethanol series (90, 85, 80, 75, 70, 50, 25 and 0%, diluted with sterile PBS). At each step, incubate at 4 °C until the scaffolds have sunk to the bottom of the vessel before changing the solution. Degas the scaffolds under light vacuum if bubble formation within the scaffolds is observed.
   5. Replace the sterile PBS for an additional two rinses to remove residual ethanol.
   6. Store in 100% sterile PBS at 4 °C until ready for cell culture. Use the scaffolds within 1 – 2 weeks following rehydration.
2. Preparation for Cell Seeding
   1. On the day prior to seeding, transfer the foams using forceps into cell culture treated well plates or transwell inserts and add sufficient cell culture medium (selected based on the cell type of interest) to completely submerge the scaffolds (e.g., 2.5 mL/scaffold in a 12-well plate). Similarly, allow the microcarriers to gravity settle within the centrifuge tube, carefully remove the PBS using a serological pipette without disturbing the microcarriers, and add cell culture medium to achieve a 5:1 ratio of medium to the microcarriers.
      NOTE: For seeding human ASCs, utilize Dulbecco's Modified Eagle's medium (DMEM):Ham's F12 nutrient mixture supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
   2. Replace the cell culture medium for a total of one rinse.
   3. Equilibrate the scaffolds overnight in cell culture medium at 37 °C. The scaffolds will be ready for cell seeding the next day.
      NOTE: The foams can be statically seeded in cell culture inserts or tissue culture well plates²⁹, or dynamically seeded using a laboratory shaker³⁶. Depending on the ECM source, processing methods and suspension concentration, dynamic seeding may enhance initial cell attachment, proliferation and infiltration. The microcarriers can be seeded dynamically using a spinner culture system¹¹.