Cryostor cs10

WJ-MSCs were expanded from umbilical cords obtained with written informed consent and approval by the Institutional Review Board of the University of Miami (IRB number 20100986). Wharton’s Jelly (WJ) isolated from a human umbilical cord obtained from a healthy and full-term infant was first cut into small pieces. The pieces were placed into several Petri dishes with a minimum medium to allow attachment. The dishes were placed into a 37ºC incubator with 5% CO₂. The growth medium was composed of 20% fetal bovine serum in α-MEM. When passage 1 cells reached confluence, the cells were harvested and cryopreserved at 2 to 5 × 10⁶ cells/mL of cryopreservation medium. The cryopreservation medium used was CryoStor CS10 (BioLife Solutions, Bothell, WA). Flow cytometry analysis with CD31, CD45, CD105, CD90 antibodies, and isotype control was performed on P2 WJ-MSC to confirm the MSC identity.

To obtain immune cells from whole lung, mice were deeply anesthetized by intraperitoneal injection of ketamine and xylazine⁴⁴ and lungs were excised through thoracotomy approach. Upon excision, pulmonary vasculature was not perfused in order to allow inclusion of potential intravascular immune cells in subsequent analysis. Mediastinal lymph nodes and central airways were separated from the lungs because the goal was to analyze pulmonary parenchyma. All lobes of the right lung were then subjected to gentle tissue digestion at room temperature for 10 minutes with Liberase TL enzyme (Roche) and mechanically dissociated through a 40-micron sieve to yield a single-cell suspension (~10⁶ cells). Cells were cryopreserved in Cryostor CS10 (BioLife Solutions, Bothell, WA) following the manufacturer’s protocol to be used for subsequent staining with CyTOF-ready antibodies^{23 (link)}. Bronchoalveolar fluid was obtained from mice, which were first euthanized by CO₂ inhalation. Lack of respiratory and cardiac activity was confirmed and 22 G i.v. cannula was inserted into trachea via neck incision. Lungs were lavaged three times with 1 mL of phosphate buffered saline, fluid collected, and cell suspension obtained, as previously described^{45 (link)}. Cells were cryopreserved as described above for subsequent CyTOF antibody staining.

After amplification, hPSCs were passaged and seeded in new L7 matrix-coated flask (Nunc™ EasYFlask™, 225 cm², Thermo Fisher Scientific) at 1 clump per cm² with StemPro^® hES SFM medium supplemented with stabilized FGF2 for one day (D0). The medium was replaced by D-KSFM^® (defined Keratinocytes Serum-Free Medium, Invitrogen, Waltham, MA, USA) supplemented with 0.273 nM BMP-4 (Peprotech, Cranbury, NJ, USA) and 1 μM retinoic acid (Sigma Aldrich) on day 1 and 3 to induce differentiation. On D6, medium was switched to D-KSFM^® alone until the end of differentiation stage (between D15 and 21). Keratinocytes derived from hPSC (KER-hPSC) were sorted by differential trypsinization (0,05% v/v; Invitrogen) and amplified in higher certified KER medium (CnT-07.HC; CELLnTEC) on medical grade collagen type I (Collagen Solutions, Eden Prairie, MN, USA) coated CellSTACK^® 5-chamber (Corning^®, Corning, NY, USA, 3180 cm²) at 30,000 cells per cm² until 100% confluency during 2 passages for the maturation stage of the process. Cells were then frozen in an animal-component-free, defined cryopreservation medium with 10% DMSO (Cryostor CS10, Biolife Solutions, Bothell, DC, USA) at the end of passage p1 (Figure 1). All quality controls were carried out after thawing of KER-hPSC at p2.

Mouse tumor samples were processed as previously described²² . Briefly, right half of the brain was separated and minced with a scalpel on ice in calcium containing 1× HBSS (GIBCO) supplemented with 1 mg/ml Collagenase IV (Sigma) and 0.25 mg/ml DNase I (Sigma) and incubated for 1 h with intermittent shaking at 37 °C. Tumor Infiltrating immune cells (TIICs) were separated by a 30 and 70% percoll gradient centrifugation.
Patient tumor pieces were minced and suspended in 1 ml of RPMI without phenol red supplemented with 1 mM GlutaMAX (Life Technologies), antibiotic–antimycotic (Life Technologies), 2 mM MEM nonessential amino acids (Life Technologies), 10 mM HEPES (Life Technologies), 2.5 × 10⁻⁵ M 2-mercaptoethanol (Sigma-Aldrich) and 200 µg/ml final Liberase TL enzyme mix (Roche). The tissue was digested for 10 min at room temperature followed by three low-speed (200 g) spins to separate the cells from debris and a total of 6.40E + 05-4.00E + 06 cells were present in each sample. The human tumor cells or mouse TIICs were resuspended in Cryostor CS10 (BioLife Solutions) for long-term cryopreservation in liquid nitrogen. Between 0.5 and 1.0 × 10⁶ cells were used for each sample post thawing.