Determination of cell proliferation in collagen type I scaffolds

Introduction

3D in vitro models offer a better representation of in vivo conditions, which is translated into differences in cellular morphology, metabolism and functions when compared with cultures in monolayer. To measure such differences, a plethora of indicators can be used; with the alamarBlue assay, levels of oxidation during respiration are detected, allowing the quantitative measure of cell viability, proliferation and cytotoxicity. The alamarBlue reagent contains the fluorescent blue indicator dye resazurin, which is reduced to resorufin when entering live cells, yielding detectable fluorescence in the red.

Materials

1. Human adipose-derived mesenchymal stem cells (hMSCs)
2. Collagen type I, porcine source
3. alamarBlue reagent
4. DMEM high glucose, GlutaMAX supplement, pyruvate
5. Fetal bovine serum
6. 100X Penicillin / Streptomycin solution
7. 25% Trypsin / 0.2% EDTA solution
8. 1X Phosphate buffered saline (without calcium/magnesium) (PBS)
9. 75 cm² cell culture flasks
10. 15 ml conical tubes
11. 1.5 ml microcentrifuge 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. Centrifuge
6. Vortex
7. Water bath
8. Vacuum pump
9. Micropipettes
10. Pipetor
11. Fluorescence plate reader

Methods

Bioink preparation:

1. Culture hMSCs in 75 cm² flasks in prewarmed 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 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.

alamarBlue assay:

10. Make a 1:10 dilution of alamarBlue reagent in complete culture media inside a conical tube. Prepare as much volume as needed. Vortex thoroughly and protect from light.
11. Wash 3D cultures 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, and only cell media) controls.
12. Add alamarBlue staining solution to each sample: use as much as needed to cover samples entirely but take into consideration that signal can be diluted when using high volumes, thus obtaining a low plate readout.
13. Place samples in the cell culture incubator (37°C, 5% CO₂) and leave incubating for the time suggested by the manufacturer (1-4 hours), protected from light. Incubation time may vary depending on scaffold composition and geometry.
14. Collect supernatants in microcentrifuge tubes, and mix well by up and down
15. Transfer 200 µl of supernatant to a well of a 96-well plate. Make triplicates.
16. Protect the plate from light and store at 4°C until reading (do not store for more than 3 days).
17. Record results using a fluorescence plate reader using excitation wavelengths between 540-570 nm and read emission within the range of 580-610 nm.
18. As alamarBlue is a non-toxic reagent, either endpoint or kinetic assays of cell metabolism, cell viability or compound toxicity can be performed.

Number	Category	Product	Amount
1	-	Human adipose-derived mesenchymal stem cells	1
2	-	Collagen type I, porcine source	1
3	-	alamarBlue reagent	1
4	-	DMEM high glucose, GlutaMAX supplement, pyruvate	1
5	-	Fetal bovine serum	1
6	-	100X Penicillin / Streptomycin solution	1
7	-	0.25% Trypsin / 0.2% EDTA solution	1
8	-	1X Phosphate buffered saline (without calcium/magnesium)	1
9	-	75 cm2 cell culture flasks	1
10	-	15 ml conical tubes	1
11	-	1.5 ml microcentrifuge 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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