Scaffold biocompatibility assessment

Introduction

To date, most cell-based assays use traditional 2D monolayer cells cultured on flat and rigid substrates. However, these models are unable to reproduce the architecture, mechanical and functional properties inherent to native tissues, thus hampering the translation of research results into clinics. For these reasons, 3D cell culture systems are increasingly gaining interest in areas such as drug discovery and tissue engineering. They provide more physiologically relevant information and more predictive data than 2D cultures. In them, cells are cultured in supportive materials termed scaffolds. One of the first steps when developing new scaffolds consists on the assessment of their biocompatibility through different bioassays.

Materials

1. Human adipose-derived mesenchymal stem cells (hMSCs)
2. Collagen type I, porcine source
3. Live/DEAD Viability/Cytotoxicity kit, for mammalian cells
4. Hoechst 33342
5. DMEM high glucose, GlutaMAX supplement, pyruvate
6. Fetal bovine serum
7. 100X Penicillin / Streptomycin solution
8. 25% Trypsin / 0.2% EDTA solution
9. 1X Phosphate buffered saline (without calcium/magnesium) (PBS)
10. 75 cm² cell culture flasks
11. 15 ml conical tubes
12. 12-well culture plates
13. 5 ml plastic syringes and 21G needles
14. Luer-to-luer connector
15. 0.41 mm ID conical tips
16. Glass Pasteur pipettes
17. Tips for micropipettes
18. Serological pipettes

Equipment required

1. REG4Life bioprinter
2. Biosafety cabinet (class II A)
3. CO₂ incubator
4. Optical microscope
5. Confocal microscope
6. Centrifuge
7. Vortex
8. Water bath
9. Vacuum pump
10. Micropipettes
11. Pipetor

Methods

Preparation of bioink:

1. Culture hMSCs in 75 cm² flasks in complete culture media (DMEM + 10% fetal bovine serum + 1X penicillin / streptomycin solution) in a cell culture incubator (37°C, 5% CO₂). Use an optical microscope for the follow-up.
2. Aspirate the media, wash with prewarmed 1X PBS, add trypsin/EDTA to cover the entire surface and incubate in the cell culture incubator until cells detach (2-3 min approx.).
3. Stop the reaction by adding complete culture media and collect the cell suspension in a 15 ml conical tube.
4. Centrifuge cells at 1,000 rpm for 5 minutes, aspirate supernatant and resuspend pellet in complete culture media (250 µl per each million of cells).
5. Transfer cell suspension in a 1.5 ml microcentrifuge tube and aspirate it through a 21G needle connected to a 5 ml syringe.
6. Mix cell suspension with neutralized collagen type I to obtain a final density of 1 million cells / ml of collagen. Use two syringes connected with a luer-to-luer connector and push plunger back and forth until obtaining a homogenized mixture.
7. Connect a 0.41 mm conical tip in the syringe containing the bioink.
8. Place the syringe in the nozzle of a REG4Life bioprinter and print the desired structure in a 12-well culture plate. 20x20x0.5 mm (w x l x h) solid square patterns were created in this case as an example.
9. Add complete culture media to cover the entire construct and leave the plate in the incubator at 37°C and 5% CO₂.

Live/Dead assay:

10. Prepare the volume required of Live/Dead + Hoechst staining solution in 1X PBS in a tube protected from light. Ensure that the final concentrations employed are: 4µM ethidium homodimer-1, 2µM calcein and 5 µg/ml Hoechst 33342. Vortex thoroughly.
11. Wash the samples with prewarmed 1X PBS twice by aspiration with the glass Pasteur pipette connected to the vacuum system. Add positive (2D culture), negative (cells treated with high concentrations of dymethilsulfoxide) and autofluorescence (scaffold without cells) controls.
12. Add Live/Dead + Hoechst staining solution to each sample: use as much as needed to cover samples entirely
13. Place samples in the cell culture incubator and leave incubating for 30 min (37°C, 5% CO₂). Incubation time may depend on reagents difussion, which is associated with the scaffold composition and geometry.
14. Discard the staining solution and wash samples 3 times with 1X PBS. Protect them from light until image acquisition.
15. Place the 12-well plate on the stage of the confocal microscope. Acquire images using configurations compatible with maximum excitation and emission wavelengths of the dyes: calcein (ex: 488 nm; em: 517 nm), ethidium homodimer-1 (ex: 543 nm; em: 646 nm), Hoechst 33342 (ex: 405 nm; em: 453 nm)
16. Cells stained with calcein (green) are alive, cells stained with ethidium homodimer-1 (red) are dead and nuclei are counterstained with Hoechst (blue).

    

Number	Category	Product	Amount
1	-	Human adipose-derived mesenchymal stem cell	1
2	-	Collagen type I, porcine source	1
3	-	Live/DEAD Viability/Cytotoxicity kit, for mammalian cells	1
4	-	Hoechst 33342	1
5	-	DMEM high glucose, GlutaMAX supplement, pyruvate	1
6	-	Fetal bovine serum	1
7	-	100X Penicillin / Streptomycin solution	1
8	-	0.25% Trypsin / 0.2% EDTA solution	1
9	-	1X Phosphate buffered saline (without calcium/magnesium)	1
10	-	75 cm2 cell culture flasks	1
11	-	15 ml conical tubes	1
12	-	12-well culture plates	1
13	-	5 ml plastic syringes and 21G needles	1
14	-	Luer-to-luer connector	1
15	-	0.41 mm ID conical tips	1
16	-	Glass Pasteur pipettes	1
17	-	Tips for micropipettes	1
18	-	Serological pipettes	1

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