組織特異的細胞培養および配信プラットフォームとして、細胞外マトリックス由来のフォームおよびマイクロキャリアの作製

The overall goal of this protocol assisting to the size non-chemically cross linked foams or micro carriers derived from tissue specific extra cellular matrix for applications in 3D invitro cell culture models, or as pro-regenerative scaffolds in cell delivery systems. This method can help answer key questions in the fields of applied cell biology and tissue engineering such as how the 3D cellular micro environment mediates cell function and tissue regeneration. The main advantage of this technique is it enables the fabrication of scaffolds with tunable geometries that mimic the native tissue specific ECM composition and are stable in long term culture without additional cross linking.

After lyophilizing the tissue derived ECM according to the text protocol, use sharp surgical scissors to finely mince the decellularized tissue. To cryomill the processed tissue, fill a 25 milliliter stainless steel milling chamber for a laboratory ball mill system with minced lyophilized ECM, and add two 10 milliliter stainless steel milling balls. Close and completely submerge the loaded milling chamber in liquid nitrogen for three minutes.

Then mill the frozen sample at 30 Hertz for three minutes. Repeat the submersion in liquid nitrogen and the milling until the ECM is milled into a fine powder. Weigh out 250 milligrams of either minced or cryomilled ECM and transfer it to a 15 milliliter centrifuge tube.

Add five milliliters of 22 molar NaH2PO4 buffer to the centrifuge tube and mark the liquid level on the tube. Then prepare an alpha amylase stock solution by adding 7.5 milligrams of alpha amylase to one milliliter of 22 molar NaH2PO4 buffer. Next, add 100 microliters of the alpha amylase stock solution to the sample.

Then add 22 molar NaH2PO4 to obtain a final volume of 10 milliliters. Agitate the suspension continuously at 300 RPM and room temperature for 72 hours. Centrifuge the suspension at 1500 times G for 10 minutes.

Carefully collect and discard the supernatant without disturbing the digested ECM pellet. Then use 10 milliliters of five percent NaCl diluted in double distilled water to re-suspend the pelleted material and agitate continuously for 10 minutes at 300 RPM. Repeat the wash two more times before using 10 milliliters of double distilled water to re-suspend the pellet.

Then agitate the suspension continuously at 300 RPM at room temperature for 10 minutes. After centrifuging the sample again, carefully collect and discard the supernatant without disturbing the digested ECM pellet. Add 2 molar acetic acid to the five milliliter mark.

And vortex. Then continuously agitate the suspension at 120 RPM and 37 degrees Celsius overnight. The following day, using a hand held homogenizer equipped with a 10 millimeter wide saw tooth probe, homogenize the ECM suspension at room temperature in 10 second intervals until no visible fragments remain.

Place the suspension in a beaker of cold water between the homogenization intervals to prevent overheating. Incubate the ECM suspension at 37 degrees Celsius with continuous agitation at 120 RPM until the suspension is warm. Then us 2 molar acetic acid to dilute the sample suspension to the desired concentration.

With a three milliliter syringe and an 18 gauge needle, fill the desired mold with the ECM suspension. Foams can be fabricated in a range of geometries depending on the shape of the mold. Cover and freeze the molds at minus 20 or minus 80 degrees Celsius.

Then place the molds into a lyophilizer flask. Connect the flask to the laboratory freeze drier system and dry the molds for 24 hours or until fully dry. Incubate the cryomilled ECM suspension with continuous agitation at 100 RPM overnight.

Load three milliliters of ECM suspension into a three milliliter luer lock syringe and attach a winged infusion set onto the bore of the syringe. Secure the syringe within the syringe pump. Then fasten the needle to a retort stand and position the needle tip vertically at a distance of four to six centimeters from the top of a low formed Dewar flask.

Next attach an alligator clip electrode to the tip of the needle, also ensuring the alligator clip is connected to the positive terminal of the high voltage power supply. Fold a strip of aluminum foil over the edge of the vacuum flask. Then attach a second alligator clip electrode to the outer edge of the foil.

Connect it to the ground source terminal of the power supply. Fill the Dewar flask with liquid nitrogen to approximately one centimeter from the top so that three quarters of the foil is submerged. Then set the syringe pump to an infusion rate of 30 milliliters per hour and electro spray the samples according to the text protocol.

Once the infusion is complete, carefully pour off excess liquid nitrogen from the Dewar, leaving the micro carrier suspended in approximately 25 milliliters of liquid nitrogen to ensure they remain frozen. Immediately transfer the micro carriers with liquid nitrogen into a 50 milliliter centrifuged tube by pouring in one smooth motion. Then use a Scoopula to collect any frozen micro carriers remaining in the Dewar.

Add them to the centrifuge too. To prepare for lyophilization, cover the centrifuge tube with aluminum foil perforated with small holes. Place the covered centrifuge tubes into a lyophilizer flask.

Then immediately connect the flask to the lyophilizer and dry the samples overnight. Gently transfer the lyophilized foam into a 50 milliliter centrifuge tube containing excess absolute ethanol. Similarly, re-suspend the lyophilized micro carriers in excess absolute ethanol within the original centrifuge tube used for collection.

Using a Serological pipette, filter the micro carriers through a stainless sieve with a defined mesh size into a new 50 milliliter centrifuge tube to remove any aggregates and select for desired size ranges. Incubate the sample at four degrees Celsius for four hours or until the foam or micro carriers have settled to the bottom of the centrifuge tube. Then, under a laminar flow hood with aseptic technique, use a Serological pipette to remove the absolute ethanol from the scaffolds and add excess 95%ethanol diluted with sterile PBS.

Incubate the scaffolds at four degrees Celsius for four hours or until the scaffolds have sunk to the bottom of the rehydration vessel. Gradually rehydrate the scaffolds through an ethanol series. At each step, incubate the scaffolds at four degrees Celsius until they sink to the bottom of the vessel, before changing the solution.

Replace the sterile PBS with an additional two rinses to remove residual ethanol. Store the samples in 100%sterile PBS at four degrees Celsius until ready for cell culture. Use the scaffolds within one to two weeks following rehydration.

Shown here are examples of the types of ECM derived foams and micro carriers fabricated using decellularized adipose tissue. Porcine decellularized dermal tissue, and porcine decellularized left ventricle. Demonstrating the techniques can be applied to generate tissue specific bio-scaffolds using a variety of decellularized tissues as ECM sources.

Following cryomilling or mincing, enzymatic digestion, and homogenization, the samples can be dispensed into a bowl before freezing and lyophilization. This figure represents rehydrated DAT, DDT, and DLV milled foams, synthesized in a 48 well tissue culture plate mold. These DAT, DDT, and DLV foams were fabricated with cryomilled ECM at a concentration of 35 milligrams per milliliter and a freezing temperature of minus 80 degrees Celsius.

To fabricate ECM derived micro carriers using an electro spraying technique, for each ECM source suspension concentration, needle gauge infusion rate, and voltage can be tuned to generate discrete spherical micro carriers ranging from 350 to 500 micrometers in diameter following controlled rehydration. Finally, the DAT, DDT, and DLV micro carriers shown here by SEM reveal that the ultra structure in size can vary depending on the de-cellularized ECM source. The methods presented in this video can be used to fabricate a diverse array of tissue specific micro carriers and foams comprised of pure, non-chemically cross linked ECM using de-cellularized tissue as a matrix source.

Following these procedures, the foams and micro carriers can be seeded with cells under static or dynamic conditions and can be applied as a cell instructive substrate for invitro cell culture and invivo applications.