Bioreactor Processed Stromal Cell Seeding and Cultivation on Decellularized Pericardium Patches for Cardiovascular Use

Abstract

(1) Background: Decellularized xenogeneic tissues are promising matrices for developing tissue-engineered cardiovascular grafts. In vitro recellularization of these tissues with stromal cells can provide a better in vivo remodelling and a lower thrombogenicity of the graft. The process of recellularization can be accelerated using a cultivation bioreactor simulating physiological conditions and stimuli. (2) Methods: Porcine pericardium was decellularized using a custom-built decellularization system with an optimized protocol. Autologous porcine adipose-derived stromal cells (PrASCs), isolated from the subcutaneous fat tissue, were used for recellularizing the decellularized pericardium. A custom cultivation bioreactor allowing the fixing of the decellularized tissue into a special cultivation chamber was created. The bioreactor maintained micro-perfusion and pulsatile pressure stimulation in order to promote the ingrowth of PrASCs inside the tissue and their differentiation. (3) Results: The dynamic cultivation promoted the ingrowth of cells into the decellularized tissue. Under static conditions, the cells penetrated only to the depth of 50 µm, whereas under dynamic conditions, the tissue was colonized up to 250 µm. The dynamic cultivation also supported the cell differentiation towards smooth muscle cells (SMCs). In order to ensure homogeneous cell colonization of the decellularized matrices, the bioreactor was designed to allow seeding of the cells from both sides of the tissue prior to the stimulation. In this case, the decellularized tissue was recolonized with cells within 5 days of dynamic cultivation. (4) Conclusions: Our newly designed dynamic bioreactor markedly accelerated the colonization of decellularized pericardium with ASCs and cell differentiation towards the SMC phenotype.

Collagen Bioinks for Bioprinting: A Systematic Review of Hydrogel Properties, Bioprinting Parameters, Protocols, and Bioprinted Structure Characteristics

“…The satisfactory cell growth and good viability were then observed even in high concentrated gels (>10 mg/mL) when porcine stromal stem cells derived from adipose tissue were cultivated under static conditions. The protocol of harvesting and their characterisation is described more in our study [ 82 ]. Hydrogels were prepared in syringes and 3D printed onto coverslip glass.…”

Growth Factors VEGF-A165 and FGF-2 as Multifunctional Biomolecules Governing Cell Adhesion and Proliferation

“…This stimulation can also be substituted by electrical, magnetic, gravity, or ultrasound stimulation, and is important for proper cell differentiation and phenotypic maturation. In addition, the media flow in dynamic cell culture systems enables a better supply of cells with oxygen and nutrients and quick waste removal, which further improves the physiological functions of cells [ 69 , 70 ]. Dynamic cultivation is, therefore, indispensable in advanced tissue engineering, which aims to create replacements of damaged tissues, closely mimicking the well-functioning tissues in a healthy organism.…”

“…Porcine adipose tissue-derived stem cells (PrADSCs) were isolated from fat surgically extracted from the neck area of experimental pigs (breed Prestice black pied pigs with a weight of approximately 35–40 kg; Institute of Animal Science, Přeštice, Czech Republic) during the surgery under general anesthesia. The protocol for cell isolation was also set according to Estes et al [ 49 ] with some modifications developed in studies focused on adipose-derived stem or stromal cells [ 74 , 75 ] and is described in our previous study by Matejka et al, 2020 [ 70 ]. The cells were expanded in a standard way until the 2nd passage in DMEM-F12K (Sigma-Aldrich, St. Louis, MO, USA) medium (ratio 1:1) supplemented with 10% of FBS (Sigma-Aldrich, St. Louis, MO, USA), 1% of ABAM (Antibiotic Antimycotic Solution, contains 100 units penicillin, 0.1 mg streptomycin, and 0.25 µg amphotericin B per mL of culture media, Sigma-Aldrich, St. Louis, MO, USA) and 10 ng/mL of FGF-2 (GenScript, Piscataway, NJ, USA, Cat.…”

“…CD73 was present only in 0.3–2.6% of the cells but is known from the literature that this marker is very low or absent in porcine ADSCs, and instead of it, CD44, i.e., hyaluronan receptor, has been usually evaluated. The prADSCs were almost negative for CD34 (0.5–1%) and CD45 (1–3%) but they showed a relatively high positivity for CD31 (29–35%) [ 70 ].…”

pH Modification of High-Concentrated Collagen Bioinks as a Factor Affecting Cell Viability, Mechanical Properties, and Printability

“…A cell suspension was prepared from porcine stromal cells derived from porcine adipose tissue isolated from the fat of the neck area. The whole procedure, including cell characterization, is described in a previous article by Matejka et al [ 56 ]. Cells were pre-cultured in the growth medium described above in 75 cm 2 culture flasks (TPP, Trasadingen, Switzerland).…”