Page 310 - IJB-10-6

International Journal of Bioprinting Bioprinted plasma biocarriers for MSC delivery

collected after 96 h in a non-inflamed environment in vitro. transferred to a bioprinter head maintained at 22°C for
Understanding how inflammatory molecules influence at least 5 min to ensure uniform temperature throughout
MSC activities is crucial, given that MSCs frequently the cartridge. The BioX printer was operated under the
encounter an inflammatory milieu within host tissues. following conditions: an extrusion pressure of 30–50 kPa,
Without a suitable delivery vehicle, making therapeutic a 20G plastic conical tip, a cartridge maintained at 22ºC, a
projections is challenging due to uncertainties regarding pre-flow of −300 ms, a head movement speed of 5 mm/s,
cell survival in the joint. Therefore, we created reproducible and a bed temperature of 10ºC. Following bioprinting, each
BMSC biocarriers through bioprinting and examined their structure was photocured using the BioX 405 photocuring
main properties and trophic actions in vitro. module at 100% power, 4 cm height, for 60 s.
2. Methods 2.4. MSC viability within bioprinted biocarriers
The viability of MSCs was assessed using the CyQUANT
2.1. Preparation of plasma products TN XTT Cell Viability Assay (Invitrogen, Thermo Fisher,
Whole blood anticoagulated with citrate phosphate USA). GelMA bioinks mixed with an MSC suspension were
dextrose (CPD) was collected from six healthy donors photocrosslinked and incubated. After adding the XTT
via apheresis and processed by the Basque Biobank. working solution and further incubation, absorbances at
Leuko-reduced PRP and fresh frozen plasma (FFP) were 660 and 450 nm were measured to calculate the specific
generated using the REVEOS Automated Blood Processing absorbance. Experiments were conducted in triplicate.
System (TERUMO BCT, Japan). PRP samples were diluted
1:5 in FFP to standardize platelet counts. Due to limited 2.5. In vitro evaluation of biocarrier biology
blood availability at the Basque Blood Biobank, platelet with/without inflammation
donors were not matched with FFP donors. Supernatants The GelMA biocarriers enriched with PRP or FFP and
were prepared by adding 10% CaCl , incubating at 37ºC loaded with BMSCs were evaluated in non-inflamed and
2
for 1 h, then centrifuging, filtering, aliquoting, and storing inflamed environments (50 ng/mL IL-1β or 50 ng/mL
at −80ºC. TNF-α). The conditioned medium was collected after 96
h and centrifuged, and the supernatants were stored at
2.2. Preparation of functionalized GelMA −80ºC for further analysis.
To prepare the GelMA stock solution, sterile lyophilized
GelMA (CellInk PhotoGel® 50% DS; CellInk, Sweden) was 2.6. Antibody-based protein arrays
combined with sterile-filtered Dulbecco’s Modified Eagle Protein release from BMSCs in various hydrogel
Medium/Nutrient Mixture F12 (DMEM-F12; ThermoFisher formulations was evaluated using the Human Cytokine
Scientific Inc., USA), supplemented with 0.5% (w/v) Array Q1000 Kit (126QAH-CAA-1000-1; RayBiotech
lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) Inc., USA). Array experiments were conducted as per the
to achieve a final concentration of 10%. The solution was manufacturer’s instructions, with results quantified relative
kept at 37ºC overnight, with gentle agitation and protection to positive controls. Plasma samples from six donors were
from light, allowing it to fully dissolve without trapping air pooled to address biological variability.
bubbles. Once completely dissolved, the GelMA stock was
stored in the dark at 4ºC and used within 1 month. 2.7. Bioinformatic analyses
For bioink preparation, the GelMA stock was heated 2.7.1. STRING database
in a culture oven at 37°C for at least 30 min until liquefied. We used the STRING database (version 11.5; string-db.
It was then diluted to a 5% final concentration by mixing org, accessed on February 2024) to explore known protein
equal volumes of either preheated DMEM-F12 or the interactions and enriched biological functions. Proteins
extruded supernatant from PRP clots, all kept at 37°C. 19 overexpressed more than two-fold in PRP biocarriers
compared to nude biocarriers were selected, with a high
2.3. Bioprinting of MSC biocarriers edge confidence of 0.9. Figure 1A and B illustrates robust
Biocarriers were fabricated using a CellInk BioX bioprinter networks with edges representing physical and functional
(CellInk, Sweden). To prepare the bioink, 10% GelMA associations, excluding disconnected nodes. The main
stock was mixed with the appropriate supplement (DMEM network was clustered into four groups using k-means.
or PRP-derived secretome) and a cell concentrate (3 × Enriched functions are presented against –LOG10 (FDR),
10 MSCs/mL) in a 50:40:10 ratio. All components were where FDR represents the false discovery rate.
6
pre-warmed to 37°C before mixing. The prepared bioinks
were then loaded into 3 mL pneumatic syringes and briefly 2.7.2. Ingenuity pathway analysis
placed in a freezer for 2 min to accelerate gelation and Proteins secreted by MSCs were categorized based on Gene
prevent cell precipitation. After gelation, the syringes were Ontology (GO) functions. We compared PRP biocarriers

Volume 10 Issue 6 (2024) 302 doi: 10.36922/ijb.4426