Versene solution

The MEG-01 (human megakaryoblastic cell line, ATCC) and THP-01 (human monocytic cell line from the collection of Moscow State University https://human.depo.msu.ru, accessed on 3 September 2018) cell lines were cultured in RPMI-1640 complete growth medium (Thermo Fisher, Carlsbad, CA, USA) containing an antibiotic-antimycotic (HyClone, Marlborough, MA, USA) and 10% fetal bovine serum (FBS, HyClone). Primary human umbilical vein endothelial cells (HUVEC, https://human.depo.msu.ru, accessed on 3 September 2018) were cultured in EBM-2 complete medium with additives (Lonza, Basel, Switzerland), an antibiotic-antimycotic (HyClone), and 2% FBS (HyClone). Chinese hamster ovary (CHO) cells were cultured in Dulbecco’s modified Eagle’s medium-F12 (HyClone or PanEco) with an antibiotic-antimycotic solution (HyClone) and 10% FBS (HyClone). All cells were passaged by treatment with the Versene solution (PanEco, Moscow, Russia) and HyQTase protease mixture (HyClone). All cell lines were cultured in an incubator at 37 °C and 5% CO₂.

Cytotoxicity of SA scaffolds has been evaluated by a colorimetric method using MTT. MSCs were detached from the surface of Petri dishes using a Versene solution (PanEco, Russia) with the addition of 0.25% trypsin (PanEco, Russia) and seeded in 24-well plates (Corning, NY, USA) with a density of 5 × 10⁴ cells per well. Non-cross-linked SA, control scaffolds cross-linked with 2 wt.% or 10 wt.% CC solutions, as well as gene-activated scaffolds, were placed in Transwell system with a pore size of 8 µm (Corning Transwell). The wells without scaffolds (cells only) were used as controls. After 1 and 7 days, MTT (tetrazolium (3-(4,5-dimethyl thiazolyl−2)−2,5-diphenyltetrazolium bromide, PanEco, Russia) at a concentration of 0.5 mg/mL was added to the wells and incubated for 2 h at 37 °C. Formazan crystals were extracted from cells using dimethylsulfoxide (DMSO, PanEco, Russia) stirring on a shaker for 20 min. The formazan absorption was evaluated by measuring the optical density of the eluate at a wavelength of 570 nm subtracting the background value at 620 nm on a BioRad Reader XMark tablet reader (Bio-Rad Laboratories, Hercules, CA, USA).

For immunophenotyping of SCs, the MSC Phenotyping Kit (130-095-198, Miltenyi Biotec GmbH, Germany) and antibody to CD31 (303124, Biolegend, United States) with corresponding control (400158, Biolegend, United States) were used according to the manufacturer’s instructions. Cells were washed with Versene solution (Paneco, Russia), detached from plastic with 0.05% trypsin-EDTA solution (Gibco, United States), and after trypsin inhibition by complete growth medium, they were aliquoted in four test tubes and centrifuged for 10 min at 300 g. Pellets were then resuspended in flow cytometry buffer [1% bovine serum albumin (BSA; Sigma, United States) in HBSS (Paneco, Russia)] and incubated for 10 min at 4°C with antibodies to CD31 or cocktail of antibodies to CD73, CD90, CD105, CD14, CD20, CD34, CD45 from the kit or corresponding control antibodies at a dilution provided by the manufacturer. After labeling, cells were diluted by 1 ml of flow cytometry buffer, centrifuged for 10 min at 300 g, then supernatant was discarded, and cells were resuspended in 500 μl of flow cytometry buffer. Cells were detected using BD LSR Fortessa flow cytometer (BD Biosciences, United States), and results were analyzed using the FlowJo software (BD, United States).

Analysis of the scaffold biodegradation was conducted in two types of solution: phosphate buffer (PBS)—for passive biodegradation—and a 0.25% trypsin in Versene solution (PanEco, Moscow, Russia)—for enzymatic biodegradation. The analysis was performed using scaffold samples with a diameter of 8 mm (7 samples of each type of scaffold, as specified in Section 2.1). The samples were placed in 24-well plates. To each sample was added 1 mL of PBS or trypsin solution. The plates were kept under standard conditions in a CO₂-incubator (under the following conditions: +37 °C; 5% CO₂; absolute humidity) throughout the experiment. At test points (from 2 to 1008 h), 100 μL samples were taken from the wells containing the materials under study. The samples were frozen and stored at 80 °C. The volumes taken as samples were compensated by adding appropriate medium (100 μL) to the corresponding wells [51 (link)].
After the scaffold incubation had been completed, the frozen samples were examined. The amount of total protein in the samples was estimated. For this, the samples were thawed (at room temperature; 24 h) and the protein concentration determined using an IRF-456 refractometer (KARAT MT, Moscow, Russia). A calibration graph made using human serum albumin was used to determine the total protein concentration. The determination error rate was 0.02%.

To functionally examine the pluripotency of hPSC lines, their differentiation potential towards three germ layers was evaluated using embryoid body (EB) formation. In brief, undifferentiated colonies of all hPSC lines in middle passages (26–28 p) were enzymatically detached (Versene solution; Paneco) and replated in ultra-low attachment plates (Corning, Corning, NY, USA) with mTeSR™-1 medium (StemCell Technologies, Vancouver, BC, Canada) and Rock inhibitor (StemCell Technologies). The next day, cells that formed aggregates were transferred to a new ultra-low attachment plate to form EBs under differentiation medium DMEM-F12 (Invitrogen, Waltham, MA, USA), 20% (v/v) Knockout Serum Replacement (Invitrogen), 1% L-glutamine, 1% nonessential amino acids (NEAA; Invitrogen), and 1% penicillin-streptomycin (Invitrogen). After 7 days of EB cultures in suspension, EBs were transferred to a 12-well plate covered with 0.1% gelatin and were allowed to attach for further differentiation over 10 days. On day 10, the differentiated EBs were stained for germ layer markers with antibodies anti-TUJ1 (BioLegend, San Diego, CA, USA; 801213), anti-AFP (GeneTex, Irvine, CA, USA; GTX131311), and anti-alpha-SMA (Sigma Aldrich, St. Louis, MO, USA; A2547-.2ML), or cells were harvested for RNA isolation.

On the fifth day of DE differentiation, monolayer cell cultures were dissociated to single-cell suspensions with Versene solution (Paneco) for 10 min at 37 °C in 5% CO₂ and then collected by centrifugation at 300× g for 5 min. Cells were fixed with 2% paraformaldehyde for 5 min at room temperature, then centrifuged at 600× g for 5 min and permeabilized in 0.2% Triton X-100 in PBS for 5 min. After 30 min of incubation in blocking solution (2.5% BSA, 0.1% Tween 20 in PBS), primary antibodies were added for 3 h at RT. Cells were washed once with 0.1% Tween20 in PBS and subjected to incubation with appropriate secondary antibodies conjugated to FITC for 1 h at RT in the dark. Antibodies and final dilutions are given in Table S3. Flow cytometry analysis was performed using a NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA, USA). Positive cells were gated on a dot plot of FSC versus FITC fluorescence.

Primary cultures were obtained from human postoperative glioma, complying with all the formal requirements of the Russian Federation. This study was approved by the Ethics Committee of Burdenko Neurosurgical Institute, Russian Academy of Medical Sciences (No_12/2020). All subjects gave written informed consent in accordance with the Declaration of Helsinki. All cell cultures were adherent cell cultures. The cells were cultured in DMEM/F12 medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco, USA), 2 mM L-glutamic acid (Paneco, Moscow, Russia), and 1% antibiotic/antimycotic solution (penicillin/streptomycin) (Corning, Corning, NY, USA) at 37 °С and 5% СО₂. Cells were removed from culture vessels using Versene solution (Paneco, Russia) and 0.05% Trypsin solution (Gibco, USA).