Once the stem/progenitor cells formed a confluent monolayer, 2 × 105 cells were seeded on the apical chamber of a 24-well Transwell (Corning Transwell 3470) pre-coated with 30 µg/ml PureCol (Advanced BioMatrix). Cells seeded on Transwells were first cultured in submerged phase. Once the cell layer on the Transwell membrane was observed to be intact and fully confluent (2-4 days post-seeding), the medium on the apical chamber was removed and the cells were cultured at ALI thereafter. Basolateral medium was refreshed twice a week and the cells were differentiated for 18 days in ALI-Diff medium (Supplementary Table 2) and subsequently differentiated into airway organoids (AOs). To promote goblet cell differentiation, bAECs cultured for 7 days at ALI were treated with 10 ng/ml human Inteleukin-13 (IL-13, StemCell technologies) for an additional 14 days before cell infection or other assays. Frozen stocks of ALI-AECs dissociated into single cells were stored in CryoStor® CS10 cell freezing medium (StemCell technologies).
Cryostor cs10 cell freezing medium
Manufactured by STEMCELL
Sourced in Canada
CryoStor® CS10 is a cell freezing medium that is designed to protect cells during cryopreservation. It is a sterile, ready-to-use solution that contains a cryoprotective agent and other components to help maintain cell viability and function during the freezing and thawing process.
Automatically generated - may contain errors
Lab products found in correlation
Transwell 3470, by Advanced BioMatrix (1 mentions)
Purecol, by Advanced BioMatrix (1 mentions)
Cellbind surface hyperflask cell culture vessels, by Corning (1 mentions)
Stemmacs msc expansion medium, by Miltenyi Biotec (1 mentions)
Aim 5, by Thermo Fisher Scientific (1 mentions)
Human serum albumin (hsa), by CSL Behring (1 mentions)
Ficoll density gradient separation, by GE Healthcare (1 mentions)
5 protocols using cryostor cs10 cell freezing medium
1
Airway Organoid Differentiation Protocol
2
Expansion and Cryopreservation of UC-MSCs
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The selected UC-MSC lines at P4 were thawed in prewarm StemMACS™ MSC expansion medium (Miltenyi Biotec) and centrifuged at 500×g for 5 min at room temperature. The cells were then resuspended in StemMACS medium and reseeded into a T75 Nunc™ EasYFlask™ Cell Culture Flask at a density of 3200 cells/cm2. Once the culture reached 80% confluence, the cells were incubated with TrypLE Select for 4 min at 37 °C and diluted with StemMACS medium, followed by centrifugation at 400×g for 5 min. The cells were counted and seeded into Corning® CellBIND® Surface HYPERFlask® cell culture vessels at a density of 3400 cells/cm2. When the culture reached 80% confluence, the cells were harvested for further analysis and cryopreserved in CryoStor CS10 cell freezing medium (Stemcell Technologies, Canada), followed by storage in liquid nitrogen for further use.
3
Isolation and Culture of Bat Tracheal Cells
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E. spelaea bats used in this study were part of a captive breeding colony and handled according to protocols approved by the SingHealth Institutional Animal Use and Ethics Committee (IACUC number 2020/SHS/1582). Tracheal tissue were cut into approximately 1 mm3 pieces and rinsed with Hank's Balanced Salt Solution (HBSS) supplemented with 2.5 µg/mL amphotericin B, 50 µg/ml gentamicin and 2% P/S. Tissue fragments were incubated with 20 mg/ml protease type XIV in RPMI 1640 supplemented with 2.5 µg/mL amphotericin B, 50 µg/ml gentamicin, and 2% P/S overnight with rotation at 4°C. Digested tissues were filtered through a 100 µM cell strainer and cell clumps retained on the strainer were dissociated with a syringe plunger. Cells were centrifuged and the pellet was resuspended in RPMI 1640 with 10% FBS to inhibit digestion. Cells were washed with RPMI 1640 twice and plated on human collagen IV pre-coated 6-well plate and cultured in B/D expansion medium (Supplementary Table 1) to grow as a monolayer. Medium was refreshed every two days until confluent. Undifferentiated cells derived from digestion of primary tissue were also stored as frozen stocks in CryoStor® CS10 cell freezing medium (StemCell technologies) for future use.
4
Airway Organoid Expansion and Differentiation
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ALI-AECs differentiated for 18 days were detached from Transwells with TrypLE and resuspended in Matrigel culturing in Airway Organoid (AO) medium (Supplementary Table 3) supplemented with 25% R-spondin-1 conditioned medium, 25 ng/ml FGF7 and 100 ng/ml FGF10 for 3D expansion. Cells were expanded and formed spheroid structures in Matrigel. At day 5 of expansion, spheroids were passaged onto the apical chamber of a 12-well insert for differentiation at ALI for an additional 4–6 weeks. Medium was refreshed twice a week using AO medium with 5 ng/ml FGF7 and 20 ng/ml FGF10 until the organoids were well-differentiated. Once AOs were mature, they were frozen in CryoStor® CS10 cell freezing medium (StemCell technologies) for long-term storage.
5
Expansion and Cryopreservation of T Cells
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PBMCs were isolated from leukapheresis units using Ficoll density gradient separation (GE Healthcare, Chicago, IL, USA) and cryopreserved. PBMCs were thawed and cultured in AIM-V (Thermo Fisher Scientific, Waltham, MA) supplemented with 5% CTSTM Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific, Waltham, MA) and human IL-2 (300 IU/ml, Quangang, Shandong, China). PBMCs were activated and expanded with CTSTM DynabeadsTM CD3/CD28 (Thermo Fisher Scientific, Waltham, MA) for 24 h. T cells were transduced with LVs at multiplicity of infection (MOI) of 1. Three days after transduction, the cells were extensively washed to remove the LV particles. T cells were further expanded in AIM-V medium supplemented with 5% CTSTM Immune Cell SR (Thermo Fisher Scientific, Waltham, MA) for three to ten days in the presence of IL-2 (300 IU/ml, Quangang). The cell product was washed twice with normal saline (Qidu pharmaceuticals, Shandong, China) containing 2.5 % human serum albumin (CSL Behring, King of Prussia, PA, USA). Cells were resuspended in Cryostor® CS10 Cell Freezing Medium (STEMCELL Technologies, Vancouver, Canada) for cryopreservation and stored in liquid nitrogen.
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