A Novel Protocol to Generate Decellularized Bovine Spinal Cord Extracellular Matrix-based Scaffolds (3D-dCBS)

Materials and Reagents

1. 48-well Clear TC-treated Multiple Well Plates, Individually Wrapped, Sterile (Corning Incorporated, Costar^®, catalog number: 3548)
2. Falcon, 50 mI conical centrifuge tubes (Isolab, sterile, catalog number: 078.02.008)
3. Falcon, 15 mI conical centrifuge tubes (Isolab, sterile, catalog number: 078.02.007)
4. Borosilicate laboratory bottle, 250 mI, GL-45 (Interlab, catalog number: 061.01.250)
5. Borosilicate laboratory bottle, 500 mI, GL-45 (Interlab, catalog number: 061.01.500)
6. Metal pot, stainless steel, 250 mm diameter and 85 mm depth
7. Scalpel, length 25 mm (Interlab, catalog number: 048.51.010)
8. Scalpel blade holder, length 130 mm (Interlab, catalog number: 048.50.001)
9. Glass petri dish, diameter 60 mm; height 15 mm (Interlab, catalog number: 081.01.060)
10. Parafilm M (Sigma-Aldrich, catalog number: P7543)
11. Magnetic stirring bar; length 10 mm; diameter 6 mm (Interlab, catalog number: 057.01.010)
12. Finnpipette^TM F2 Fixed Volume Single-Channel Pipettes; 100 to 1,000 μl (Thermo-Scientific, catalog number: 4642090)
13. Finnpipette^TM F2 Fixed Volume Single-Channel Pipettes; 10 to 100 μl (Thermo-Scientific, catalog number: 4642070)
14. Glass Erlenmeyer flask; volume 50 mI (Interlab, catalog number: 027.01.050)
15. Erlenmeyer flask cap, NS 12/21 (Interlab, catalog number: 051.08.012)
16. Screwcap GL-45, blue (Interlab, catalog number: 051.09.45B)
17. Laboratory forceps, length 115 mm, 304 stainless steel (Interlab, catalog number: 048.08.115)
18. Spoon spatula, length 150 mm, 304 stainless steel (Interlab, catalog number: 047.01.150)
19. Borosilicate graduated cylinder, 250 mI (Interlab, catalog number: 015.01.250)
20. Borosilicate graduated cylinder, 500 mI (Interlab, catalog number: 015.01.500)
21. μDrop^TM Plate (Thermo-Scientific, Multiskan^TM with mdrop^TM plate)
22. BSC, the bovine spinal cord (BSC) from Holstein-Friesian cattle [thoracic region (T2–T11)] was obtained within 4-6 h following the slaughtering of healthy animals (16-month-old) at a local slaughterhouse in Balikesir, Turkey
23. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-Na₂) (Sigma-Aldrich, catalog number: E5134)
24. Trizma^® hydrochloride (Sigma-Aldrich, catalog number: T5941)
25. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4509)
26. Peracetic acid solution (Sigma-Aldrich, catalog number: 433241)
27. Ethanol 99% (Sigma-Aldrich, catalog number: 32221)
28. Magnesium chloride (Sigma-Aldrich, catalog number: M8266)
29. Deoxyribonuclease I from bovine pancreas, DNase I (Sigma-Aldrich, catalog number: DN25)
30. Ribonuclease A from bovine pancreas, RNase A (Sigma-Aldrich, catalog number: R5503)
31. Sodium hydroxide (Sigma-Aldrich, catalog number: 06203)
32. Hydrochloric acid (Sigma-Aldrich, catalog number: 07102)
33. 2-(N-Morpholino) ethanesulfonic acid (MES) (Sigma-Aldrich, catalog number: M3885)
34. N-Hydroxysuccinimide (NHS) (Sigma-Aldrich, catalog number: 8.04518)
35. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (Sigma-Aldrich, catalog number: 8.00907)
36. Milli-Q water (Merck-Millipore, Type I-III, catalog number: ZRQSVP3WW)
37. 0.1 N NaOH solution (see Recipes)
38. 1 M NaOH solution (see Recipes)
39. 1 M HCl solution (see Recipes)
40. 70% ethanol solution (see Recipes)
41. 0.05 M 2-(N-Morpholino) ethanesulfonic acid (MES) buffer (pH 5.5) (see Recipes)
42. Crosslinking (EDC/NHS) solution (see Recipes)
43. Hypertonic solution (see Recipes)
44. RNase A and DNase I enzyme solutions (see Recipes)
45. Decontamination solution (4% ethanol/0.1% peracetic acid) (see Recipes)
    

Equipment

1. Ultrasonic bath (Elma, Elmasonic S30H)
2. Lyophilizer -55 °C (Telstar, LyoQuest)
3. Milli-Q^® Integral water purification system for ultrapure water (Merck-Millipore, Type I, Direct-Q^® 3 UV)
4. Freeze dryer (Telstar, LyoQuest -55)
5. Vortex (IKA Genious 4, V4B)
6. Analytical balance (Shimadzu, model: ATX224)
7. pH Meter (Mettler Toledo, model: FiveEasy^TMFE20)
8. Magnetic stirrer (Multi Position) (Jeiotech, MS-12BB, catalog number: AAH330115BK)
9. Mechanical homogenizer (IKA, T18 Basic Ultra TURRAX)
10. Automatic single-channel pipettes, 10-100 and 100-1,000 µl (Gilson-compatible, Diamond^® Eco-Pack^TM)
11. Refrigerator (Siemens, model: KG57NPW24N)
12. Ultra-low temperature freezer (Panasonic, model: MDF-U5386S-PE)
13. Orbital shaker incubator-temperature controlled (Benchmark, model: Incu-Shaker^TM Mini)
    

Procedure

1. Dissection of BSC
   Note: Carry out the dissection process in a fume hood safety cabinet under sanitary conditions to minimize cross-contaminations.
   
   1. Harvest BSC from the thoracic region (T2-T11) of Holstein-Friesian cattle.
   2. Cut muscle, fat, and other meningeal tissues from the spinal cord using a scalpel.
   3. Rinse the tissue 3 times each with 200 ml of Milli-Q water in order to decrease connective tissue and blood contamination.
   4. After removing soft tissues, cut the spinal cord into several pieces on a glass petri dish (see Figure 1, Step 1).
      
      
      
2. Producing strategy of viscous gel from BSC
   Notes: 
   1. Carry out the tissue homogenization process at room temperature (RT) in an ice bath (prepared in a metal pot) to prevent over-heating of the mixture and the degradation of biomolecules. 
   2. Homogenization step (Step B4) should be repeated at least three times.
   1. Mince the BSC tissue (purged from external contaminants) in small pieces of approximately 2 x 2 cm dimensions. 
   2. Place BSC tissues into a glass Erlenmeyer flask using surgical equipment such as laboratory forceps made of nickel-plated steel, and stainless-steel micro spoon spatula.
   3. Weigh small pieces of BSC (approx. 5 g and in the wet form) using an analytical balance, and then place into 20 ml of 0.1 M NaOH solution.
   4. Homogenize BSC tissue pieces in 20 ml of 0.1 M NaOH solution using a homogenizer for 5 min at 10,000 rpm.
   5. Adjust the final volume of the gel solution to 30 ml with 0.1 M NaOH solution using a glass graduated measuring cylinder (see Figure 1, Step 2).
      
      
      
3. Fabrication of 3D-CBS (Figure 1)
   
   
   Figure 1. 3D-dCBS fabrication process
   
   
   1. Pipette 200 μl of the gel solution into each well of a 48-well plate to prepare 3-dimensional (3D) bovine spinal cord extracellular matrix-based scaffolds (referred to as 3D-CBS).
   2. Incubate the plate at 4 °C for 4 h for the maturation of the gel solution.
   3. Then, freeze the 48-well plate in an ultra-low temperature freezer at -80 °C overnight.
   4. Finally, freeze-dry homogenized BSC matrix overnight using a -55 °C lyophilizer (see Figure 1, Steps 3 and 4).
      
      
      
4. Crosslinking process of 3D-CBS (Figure 1)
   1. Weigh 25 mg of lyophilized 3D-CBS to prepare crosslinked scaffolds.
   2. Place each 3D-CBS into a 15 ml falcon tube including 6 ml of ethanol 99.9% at RT for 15 min while gently shaking at 120 rpm using orbital shaker incubator. 
   3. Carefully decant the ethanol solution and crosslink each 25 mg of 3D-CBS with 5.37 ml of crosslinking (EDC/NHS) solution at RT for 10 min using the orbital shaker incubator.
   4. Afterward, rinse the scaffolds with 200 ml of Milli-Q water at RT for 2 h in a 250 ml borosilicate laboratory bottle with screw cap.
   5. Finally, freeze crosslinked 3D-CBS in a 48-well plate at -80 °C and freeze-dry overnight using a -55 °C lyophilizer (see Figure 1, Step 5).
      Notes:
      1. Whereas the dimensions of non-crosslinked 3D-CBS are h=3 mm and Ø=8, the crosslinked ones are h=1.2 mm and Ø=6.
      2. Whereas mass of non-crosslinked 3D-CBS is approx. 25 mg, the crosslinked one is approx. 15 mg.
   
   
5. Decellularization process of 3D-CBS (Figure 1)
   Note: Carry out the process in the orbital shaker incubator at RT.
   1. Soak crosslinked 3D-CBS in 200 ml of hypertonic solution [50 mM Trizma^® hydrochloride, 5 mM EDTA-Na₂ (pH 8.0)] containing 1% SDS at RT for 72 h.
   2. After being treated with the hypertonic solution, rinse the scaffolds 8 times each with 250 ml of Milli-Q water for 24 h in a 250 ml borosilicate laboratory bottle with screw cap.
   3. Following the washing step, treat 3D-CBS with 50 ml of an enzyme solution containing deoxyribonuclease I (DNase I) and ribonuclease A (RNase A) at 37.5 °C for 24 h in the shaker at 120 rpm.
   4. Rinse 3D-CBS 3 times each with 250 ml of Milli-Q water and decontaminate with 200 ml of 4% ethanol and 0.1% peracetic (decontamination solution) acid for 5 h at RT. 
   5. Repeat the washing step (Step E4).
   6. Finally, freeze the scaffolds at -80 °C ultra-low temperature freezer and lyophilize overnight again to achieve 3D decellularized BSC ECM-based scaffolds (3D-dCBS) (see Figure 1, Step 6).
      Note: Mass of 3D-dCBS is approx. 2.5 mg.

Notes

1. Carefully weigh all of the materials (i.e., chemicals, reagents, tissue samples, etc.) using an analytical balance.
2. Check the pH using a benchtop pH meter that has been calibrated at the desired pH range. Use 1 M HCl or 1 M NaOH to adjust the pH of prepared solutions.
3. The storage temperature of the prepared solutions varies between 4 °C and RT. 
4. Coat falcon tubes or glass bottles with Parafilm M laboratory film to prevent drying.
5. Carry out the decellularization and washing processes in 250 mI borosilicate laboratory bottles with screw cap.
6. Figure 1 demonstrates the fabrication and decellularization processes of the 3D-CBS at a glance.
   

Recipes

1. 0.1 N NaOH solution
   1. Dissolve 0.4 g NaOH pellets in Milli-Q water so that the final volume is 100 mI
   2. Completely dissolve the pellets in the ultrasonic bath 
   3. Storage: RT; Shelf life: 3 months
2. 1 M NaOH solution
   1. Dissolve 2 g NaOH pellets in Milli-Q water so that the final volume is 50 mI
   2. Completely dissolve the pellets in the ultrasonic bath 
   3. Storage: RT; Shelf life: 3 months
3. 1 M HCl solution
   
   1. Add carefully 4.10 ml concentrated HCl (12.18 M) to 45.9 mI Milli-Q water in a fume hood
   2. Gently vortex
   3. Storage: RT; Shelf life: 3 months
   Note: Should be careful while using concentrated HCI solution.
   
4. 70% ethanol solution
   1. Mix 350 mI of ethanol (99.9%) with 150 mI of Milli-Q water
   2. Be sure to obtain a homogeneous mixture
   3. Storage: RT, Shelf life: 3 months
5. 0.05 M 2-(N-Morpholino) ethanesulfonic acid (MES) buffer (pH 5.5)
   1. Dissolve 0.54 g MES in 70% ethanol (v/v) so that the final volume is 50 mI
   2. Gently vortex for 1 min and sonicate at 280 W for 10 min
   3. Adjust the pH to 5.5 using 1 M HCl solution
   4. Storage: 4 °C; Shelf life: 1 month.
6. Crosslinking (EDC/NHS) solution
   1. For each 25 mg of 3D-CBS, dissolve 57.5 mg of N-(3-dimethylaminopropyl) N'ethylcarbodiimide hydrochloride (EDC) and 14 mg of N-hydroxysuccinimide (NHS) in 5.37 mI of MES buffer
   2. Gently sonicate at 280 W for 2 min
      
   3. Storage: 4 °C; Shelf life: prepared fresh
      Notes:
      1. Should mix EDC (2.30 g/g of sample) and NHS (0.56 g/g of sample) (molar ratio of EDC/NHS = 2.5) in MES buffer at RT.
      2. The crosslinking process previously described by Buttafoco et al. (2006) was modified and applied (215 mI of MES buffer containing EDC/NHS for each 1 g of 3D-CBS).
7. Hypertonic solution
   1. Dissolve 1.58 g of Trizma^® hydrochloride, 3.72 g of ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-Na₂) and 2 g of sodium dodecyl sulfate (SDS) in Milli-Q water
   2. Incubate at 40 °C for 10 min and then sonicate until a homogeneous solution is obtained (approx. 15 min)
   3. After being adjusted the pH 8.0 using 1 M NaOH, complete the final volume to 200 mI (referred to as the hypertonic solution)
   4. Storage: RT; Shelf life: 1 week
8. RNase A and DNase I enzyme solution
   1. Dissolve 0.19 mg of magnesium chloride (MgCl₂) and 1.58 g of Trizma^® hydrochloride in Milli-Q water using vortex
   2. After being adjusted the pH 7.5 using 1 M NaOH, complete the final volume to 200 mI (referred to as buffer solution)
   3. Add 40 mg of DNase I and 10 mg of RNase A into the buffer solution then dissolve by gentle vortexing (referred to as enzyme solution)
      Note: Should prepare as fresh and use immediately.
9. Decontamination solution (4% ethanol/0.1% peracetic acid)
   1. Mix 200 µl of peracetic acid solution (36-40%), 8 mI of ethanol (99.9%) and 191.8 mI of Milli-Q water to obtain 200 mI of decontamination solution
   2. Be sure to obtain a homogeneous mixture
   3. Storage: RT, Shelf life: 1 month

Acknowledgments

This work was financially supported by the Ministry of Science, Industry, and Technology, Republic of Turkey (Project ID. 0089.TGSD.2013).

Competing interests

The authors have declared that no competing interests exist.

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