Bioreactors with 2.3 L working volume were used in this study to cultivate Vero cells growing adherent to microcarriers (3 g/L Cytodex 1, GE Healthcare) in VP-SFM (Thermo Fisher Scientific). The pH, temperature, dissolved oxygen (DO) and stirring speed were respectively maintained at the value of 7.2, 37 °C, 50% and 90 rpm. The flow of air to the headspace of the bioreactors was controlled with a mass flow controller and maintained at 1 L/min. Cultivations were controlled by Sartorius Biostat B-DCU-3 control unit and MFCS-win software (Sartorius AG, Göttingen, Germany). All of the bioreactors were operated in batch mode and inoculated at cell density of approximately 0.1 × 106 cells/mL. The initial glucose and glutamine concentration were 35 mM and 4 mM.
Cytodex 1
Manufactured by GE Healthcare
Sourced in United States
Cytodex 1 is a microcarrier bead designed for the culture of anchorage-dependent cells. It is composed of cross-linked dextran matrix with diethylaminoethyl (DEAE) groups. Cytodex 1 provides a surface for cell attachment and growth, enabling the expansion of adherent cell lines in suspension culture.
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Lab products found in correlation
Cytodex 3, by GE Healthcare (4 mentions)
Cultispher s, by Merck Group (2 mentions)
Vp sfm, by Thermo Fisher Scientific (1 mentions)
Synthemax 2, by Corning (1 mentions)
Dmi6000b epifluorescence microscope, by Leica (1 mentions)
70 m cell strainer, by BD (1 mentions)
Accutase, by Merck Group (1 mentions)
Spectramax gemini xps, by Molecular Devices (1 mentions)
Cytopore1, by GE Healthcare (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Mtesr1, by Selleck Chemicals (1 mentions)
Y 27632, by Selleck Chemicals (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
8 protocols using cytodex 1
1
Vero Cells Cultivation on Microcarriers
2
Microcarrier Screening for eCB-MSC Attachment
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Initial microcarrier screening was performed in 6-well plates to investigate eCB-MSC attachment to five different microcarriers: Cytodex 1 (GE Healthcare Cat# 17–0448-01), Cytodex 3 (GE Healthcare Cat# 17–0485-01), Cultispher S (Sigma Cat# M9043), Enhanced Attachment (Corning Cat# 3779) and Synthemax II (Corning Cat# 3781). The cells and microcarriers were inoculated into the wells at 6700 cells/cm2 (microcarrier surface area) with 3.0 mL of 30%FBS-0bFGF medium. The 6-well plates were placed on a shaking platform (Scientific Excella e5) at 60 rpm with a ¾” diameter shaking orbit and cell attachment counts were performed at 1, 2, 3, 4 and 24 h.
3
Expansion and Infection of Vero Cells with EV71
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The expansion of Vero cells from the T-flask to spinner flasks was described previously [20 (link)]. A 3-L spinner flask containing 3 g/L microcarrier (Cytodex-1, GE) was used for Vero cell growth, with stirring at 25–40 rpm in a 37°C, 5% CO2 incubator. When the cell density reached 8×105 cells/mL, the mixture of cells and microcarriers was transferred and separated equally into four individual 1-L spinner flasks. Subsequently, the medium was replaced with fresh medium, and the cells were inoculated with ten-fold serial dilutions of EV71 B4(E59) virus stock to give MOIs of 10−1, 10−2, 10−4, or 10−6 pfu per cell. Daily sampling was performed to determine the virus titers at various time points post-infection. The endpoint of EV71 culture was the plateau of virus growth, as indicated by cytopathic effects (CPEs) greater than 90%. The virus titers were determined using a tissue culture infectious dose (TCID50) assay in Vero cells according to the Reed—Muench method [22 ].
4
Endothelial Cell Adhesion Quantification
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LECs and HUVECs were seeded to either non-coated or fibronectin-coated T25 flasks, Cytodex-1 (non-coated), fibronectin-coated Cytodex-1 or Cytodex-3 (collagen-coated) microcarrier beads (GE Life Sciences, Chalfont St Giles, GB) on day 0. IVSWT was applied on day 1 in cell culture flasks or, in case of bead stimulation, in 15 ml reaction tubes as described above. 24 h later, cells were enzymatically detached with Accutase (Sigma-Aldrich, St.Louis, USA) from flasks and beads for 7 min at 37°C. The beads were removed from cells by pipetting the cell/bead suspension through a 70 µm cell strainer (BD Falcon, Franklin Lakes, USA). The cells were centrifuged at 100×g for 5 min, counted and reseeded to non-coated 48-well plates for exact 25 min. The wells were washed twice with PBS, fixed with 4% PFA for 15 min at 4°C and nuclei were stained with 1 µg/ml DAPI in PBS/1% BSA for 1 h at 4°C. Images were taken on a Leica DMI6000B epifluorescence microscope and the amount of attached cells was quantified with ImageJ.
5
Cytodex 1 Microcarrier Preparation
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Cytodex 1 from GE HealthCare (Uppsala, Sweden) was used in this study. Microcarriers were prepared and sterilized according to the manufacturer’s instructions. Briefly, all microcarriers were hydrated in Ca2+ and Mg2+ free phosphate-buffered saline (PBS), washed twice with fresh PBS, autoclaved, and equilibrated in Leibovitz’s L15 culture medium with 10% foetal bovine serum (FBS) for at least 24 h prior to use in microcarrier-assisted culture experiments [19 (link)].
6
Microcarriers for heMSC Cultivation and Differentiation
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Cytodex 1 (GE Healthcare), Cytodex 3 (GE Healthcare), SphereCol (Advanced BioMatrix), and Cultispher-S (Sigma) microcarriers were prepared in accordance with the manufacturer’s instructions. Cytodex 1, Cytodex 3, and Cultispher-S were sterilized by autoclaving at 121 °C for 20 min while SphereCol was supplied in sterile form. All microcarriers were washed three times in MSC growth medium before use. Characteristics of the distinct microcarriers used are summarized in Table 1 .
Characteristics of microcarriers tested and their respective seeding conditions per heMSC-microcarrier construct at day 0 of differentiation
| Cytodex 1 | Cytodex 3 | SphereCol | Cultispher-S | |
|---|---|---|---|---|
| Microcarrier characteristics | ||||
| Diameter (μm) | 147–248 | 141–211 | 100–400 | 130–380 |
| Matrix | Dextran | Dextran | Type I Collagen | Gelatin |
| Charges/coating | Positively charged | Denatured collagen | – | – |
| Porosity | Microporous | Microporous | Microporous | Macroporous |
| Biodegradability | No | No | Yes | Yes |
| Surface area (cm2/mg dry weight) | 4.40 | 2.70 | – | 15.0 |
| heMSC-microcarrier construct-seeding conditions (spinner culture, data per construct) | ||||
| Cell confluency | 70% | 70% | – | – |
| Total cell number | 10.1 × 103 | 10.1 × 103 | – | – |
| Microcarrier number | 300 | 300 | – | – |
| heMSC-microcarrier construct-seeding conditions (shake flask culture, data per construct) | ||||
| Cell confluency | 70% | 70% | 70% | 70% |
| Total cell number | 10.1 × 103 | 8.88 × 103 | 8.88 × 103 | 30.8 × 103 |
| Microcarrier number | 300 | 300 | 300 | 50 |
heMSC human early mesenchymal stromal cell
7
Microcarrier-based ADSC Culture Protocols
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The microcarriers we used were cytodex 1, cytodex 3, and cytopore 1 (GE, Boston, MA, USA). The microcarrier was washed for three times with D-Hanks and stored in DMEM/F12 with 10% FBS. To generate microcarrier-based culture, an adequate amount of microcarrier was added into a non-adherent culture plate to cover the bottom of the plate. ADSCs were trypsinized and then added on to the microcarrier. This culture was established after incubation for 2 h to facilitate the cell attachment to the microcarrier with several times of mixing. To monitor the cell proliferation on the microcarriers, ADSC-EGFP cells were cultured on three types of microcarriers, and the fluorescent signals were measured by the fluorometer (SpectraMax Gemini XPS, Molecular Devices, San Jose, CA, USA). The empty microcarriers were used as background controls.
8
Microcarrier Coating for Stem Cell Culture
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Method for microcarrier coating have been reported earlier by our group [57 (link), 58 (link)]. Briefly, 20 mg/mL of Cytodex 1 (GE Healthcare, USA) was coated with (1:30 v/v) Geltrex® in DMEM/F12 medium (Thermo Fisher Scientific) overnight at 4 °C. Prior to use, pre-warmed (37 °C for 45 min) Cytodex 1 was centrifuged at 2000 rpm for 5 min. The supernatant was replaced with mTeSR™1 supplemented with 10 μM ρ-activated kinase inhibitor Y-27632 (Selleckchem, USA).
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