2. Measure the absorbance at 530 nm using a microplate reader.
3. Digest the 2-week-cultured tissue strands as well as the native
articular cartilage in the papain buffer.
4. Apply a Quant-iTTM PicoGreen dsDNA Assay Kit according
to the manufacturer’s instructions (see Note 10).
5. Measure the fluorescence intensity by a microplate reader using
a 480 nm excitation and 520 nm emission wavelength.
6. Normalize the sulfated glycosaminoglycans (sGAG) content
from each sample to dsDNA content; results are presented as
sGAG content per cell.
3.7 Total RNA
Extraction, Reverse
Transcription, and
Real-Time PCR
Analysis
1. Homogenize tissue strands in TRIzol reagent.
2. Extract total RNA using the RNeasy Mini Kit according to the
manufacturer’s instructions.
3. Reverse transcribe cDNA using TaqMan Micro RNA reverse
transcription kits according to instructions from the vendor.
4. Apply SYBR Green Real-Time PCR kit to analyze the transcription levels of cartilage-matrix-related genes, including collagen
type II, aggrecan, and chondrogenic transcription factor Sox9.
3.8 Bioprinting of
Tissue Strands
1. Load tissue strand within capsules into the detachable nozzle
(Fig. 2a).
2. Add 4% sodium citrate solution onto the capsules to decrosslink the alginate tube.
3. Remove excess sodium citrate solution from the remaining
tissue strand.
4. Mount the nozzle with tissue strand along with the mechanical
dispensing system onto the bioprinter (Fig. 2a).
5. Place a filter paper hydrated with cell culture media on the
printing platform.
6. Print the strand using printing speed at 100 mm/min and an
extrusion speed at 50.8 mm/min.
7. Load a new strand and repeat the above processes until the
entire structure is printed. Raise z axis 0.5 mm for the printing
of additive layer.
8. Hydrate the tissue strands for sufficient time to allow tissue
fusion, and transfer them to the tissue culture dish.
3.9 Self-Assembly of
Tissue Strands
1. Place multiple constructs in close proximity to each other in a
150 mm Petri dish.
2. Supplement a minimum amount of culture media to ensure
cells survive without loss of contact (Fig. 2b).
3. Assess the self-assembly of strands via imaging to monitor the
fusion process with minimal disturbance.
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Pallab Datta et al.
3. Digest the 2-week-cultured tissue strands as well as the native
articular cartilage in the papain buffer.
4. Apply a Quant-iTTM PicoGreen dsDNA Assay Kit according
to the manufacturer’s instructions (see Note 10).
5. Measure the fluorescence intensity by a microplate reader using
a 480 nm excitation and 520 nm emission wavelength.
6. Normalize the sulfated glycosaminoglycans (sGAG) content
from each sample to dsDNA content; results are presented as
sGAG content per cell.
3.7 Total RNA
Extraction, Reverse
Transcription, and
Real-Time PCR
Analysis
1. Homogenize tissue strands in TRIzol reagent.
2. Extract total RNA using the RNeasy Mini Kit according to the
manufacturer’s instructions.
3. Reverse transcribe cDNA using TaqMan Micro RNA reverse
transcription kits according to instructions from the vendor.
4. Apply SYBR Green Real-Time PCR kit to analyze the transcription levels of cartilage-matrix-related genes, including collagen
type II, aggrecan, and chondrogenic transcription factor Sox9.
3.8 Bioprinting of
Tissue Strands
1. Load tissue strand within capsules into the detachable nozzle
(Fig. 2a).
2. Add 4% sodium citrate solution onto the capsules to decrosslink the alginate tube.
3. Remove excess sodium citrate solution from the remaining
tissue strand.
4. Mount the nozzle with tissue strand along with the mechanical
dispensing system onto the bioprinter (Fig. 2a).
5. Place a filter paper hydrated with cell culture media on the
printing platform.
6. Print the strand using printing speed at 100 mm/min and an
extrusion speed at 50.8 mm/min.
7. Load a new strand and repeat the above processes until the
entire structure is printed. Raise z axis 0.5 mm for the printing
of additive layer.
8. Hydrate the tissue strands for sufficient time to allow tissue
fusion, and transfer them to the tissue culture dish.
3.9 Self-Assembly of
Tissue Strands
1. Place multiple constructs in close proximity to each other in a
150 mm Petri dish.
2. Supplement a minimum amount of culture media to ensure
cells survive without loss of contact (Fig. 2b).
3. Assess the self-assembly of strands via imaging to monitor the
fusion process with minimal disturbance.
180
Pallab Datta et al.