Page 392 - IJB-9-2

International Journal of Bioprinting In situ 3D bioprinter for skin wound healing

EDTA solution (cat.# R080), and sodium hydrocarbonate To assess the effect of platelet lysate on gel contraction
(cat.# F022E) were purchased from Paneco (Russia). and tissue spheroid spreading, it was added to the collagen
Sodium hydroxide (cat.# 1.06498) was obtained from solution at a concentration of 10%. For the preparation of
Merck (Germany). DAPI (cat.# D1306) was purchased collagen gel, 890 μL of collagen solution was mixed with
from Invitrogen (USA). 60 μL of 1 M sodium hydroxide, 250 μL of 7.5% sodium
bicarbonate, and 300 μL of PBS (for collagen gel) or 150 μL
2.7. Cell culture of PBS + 150 μL of platelet lysate (for collagen + platelet
Human umbilical vein endothelial cells (HUVECs) was lysate gel).
purchased from PromoCell (cat.# C-12203). Human
dermal fibroblasts (HFs) were obtained from Lonza (cat.# 2.10. Contraction assay
CC-2511). HUVECs were grown in a M200 medium, HF cells were harvested from confluent cultures using
supplemented with low serum growth supplement and 0.25% trypsin/0.53 mM EDTA, counted, adjusted to
antibiotic/antimycotic. HF cells were grown in DMEM the desired density, and resuspended in a solution of
containing 10% FBS. Primary cultures of rat and porcine polymerizing collagen. Aliquots (100 μL) of the cell-
fibroblasts were isolated after mechanical treatment and collagen mixture were dispensed into 96-well cell culture
disaggregation of skin samples followed by incubation plate. The hydrogel was subjected to polymerization at
with 0.25% trypsin and 200 U/ml collagenase-I solution. 37°C for 1 h, and then, culture medium was added. The
The resulting suspension was filtered and transferred to gels were then gently detached from the walls of the plate
a DMEM culture containing 10% FBS, supplemented wells by passing a medical needle around the perimeter of
with antibiotic/antimycotic and 2 mM L-glutamine. The the gels. In 48 h, brightfield images of gels were obtained
cells were incubated at 37°C in a humidified atmosphere using the “SMZ18” stereomicroscope (Nikon, Japan). The
with 5% CO and routinely split at 85%–95% confluence sample areas were measured using ImageJ 1.48v software
2
with trypsin/EDTA solution. According to DAPI staining (NIH, Bethesda, MD). All original images were converted
protocol, cells were confirmed free of mycoplasma to simplified threshold images under the same converting
contamination. condition, and the edges of the samples were manually
detected. The sample areas were first measured as pixels
2.8. Tissue spheroids formation and then converted to micrometers by comparing them to

The tissue spheroids from HUVEC and 50% HUVEC + a reference length. The results of the contraction assay were
50% HF mixture were formed using Corning spheroid expressed in percentage relative to the initial sample areas
microplates (Corning, cat.# 4520), according to the considered to be 100%.
manufacturer’s protocol. Briefly, cells in a monolayer
with 95% confluence were rinsed with EDTA solution, 2.11. Tissue spheroid spreading assay
harvested from the substrate by 0.25% trypsin/0.53 mM The tissue spheroids were formed from 3000 cells using
EDTA, and then resuspended in a cell culture medium. Corning spheroid microplates. One-day-old tissue
4
The cell concentration was 3 × 10 /mL. Finally, 100 μL of spheroids were embedded in the collagen and loaded into
cell suspensions were delivered into the wells of Corning 24-well plates (Corning, Cat.# 3337). The hydrogel was
spheroid microplates. Corning spheroid microplates subjected to polymerization at 37°C for 1 h, and then,
were incubated at 37°C in a humidified atmosphere with culture medium was added. In 48 h, spheroids were labeled
5% CO . with live/dead kit to visualize live and dead cells. Brightfield
2
and fluorescent images of spheroids were obtained
2.9. Collagen isolation from rat tails and collagen using the “Eclipse Ti-S” microscope (Nikon, Japan). The
gel preparation spreading areas and densities were measured using ImageJ

A commercial solution of “Viscoll” sterile porcine collagen 1.48v software (NIH, Bethesda, MD). The spreading areas
[31]
at a concentration of 80 mg/mL was used for in situ were expressed in percentage relative to the initial spheroid
bioprinting experiments. Before bioink preparation, the areas considered to be 100%. The densities were calculated
collagen solution was neutralized with transparent DMEM as a fraction of cells, spreading and forming spheroid, in
(without phenol red) containing TRIS buffer (pH 7.2–7.4). comparison with total image area which was consistent for
The pooled rat and porcine platelet lysate samples were all images.
prepared as described previously and the skin fibroblast
[32]
cell suspension was added to prepare the complete bioink 2.12. In vivo experiments
recipe. The resulted composition contained 10% platelet To perform in situ bioprinting experiments, 30 male
lysate and 1 million cells/mL of hydrogel solution. Wistar rats and six male Wiesenau minipigs were used.

Volume 9 Issue 2 (2023) 384 https://doi.org/10.18063/ijb.v9i2.675