4.3.2 Macromolecular crowding in ASC cultures
The macromolecular crowding method was tested for its ability to support ASC proliferation and differentiation capacity. A cocktail of macromolecules containing FicollTM400 (PM400, 17-0300-50; GE Healthcare, Bio-sciences AB) and FicollTM70 (PM70, 17-0310-50; GE Healthcare, Bio-Sciences AB) was dissolved into culture media at room temperature with gentle agitation. Fractional volume occupancy of 17% was achieved with concentrations 37.5mg/mL of FicollTM70 and 25mg/mL of FicollTM400 as described previously (C. Z. Chen et al., 2011). Media was sterile filtered after the addition of Ficoll particles.
From passage 1 onwards, the cells isolated in FBS- and HS-containing medium were divided into two populations, and the cells were expanded under +/- MMC conditions until the analyses in passage four. Due to technical reasons, the XF/SF cells were divided into two populations from passage 2 onwards, and expanded under +/-MMC conditions until the analyses in passage 4.
Table 7. Panel of cell surface markers used in all studies I-IV.
Antigen Fluorophores Surface protein Manufacturer
CD3 phycoerythrin
(PE) T-cell co-receptor BD Biosciences
CD11a allophycocyanin
(APC) Integrin alpha L (lymphocyte
function-associated antigen 1) R&D Systems Inc., Minneapolis, MN, USA CD14 phycoerythrin-
cyanine (PECy7) Lipopolysaccharide receptor BD Biosciences, Franklin Lakes, NJ, USA
CD19 PECy7 B lymphocyte-lineage
differentiation antigen BD Biosciences CD34 APC Hematopoietic progenitor cell
antigen 1 Immunotools GmbH,
Friesoythe, Germany CD45-RO APC RO isoform of leucocyte common
antigen BD Biosciences
CD54
fluorescein isothiocyanate (FITC)
Intercellular adhesion molecule 1
(ICAM-1) BD Biosciences
CD73 PE Ecto 5’ nucleotidase BD Biosciences
CD80 PE B7-1 R&D Systems Inc.
CD86 PE B7-2 R&D Systems Inc.
CD90 APC Thy-1 (T cell surface
glycoproteins) BD Biosciences
CD105 PE SH-2, endoglin R&D Systems Inc.
HLA-DR PE Major histocompatibility class II
antigen (MHC-II) Immunotools GmbH
4.4.2 Morphology of ASCs
Morphological characteristics of ASCs were observed by light microscopy to support the results of proliferation analyses. Additionally, the morphological changes during cell expansion under different culture conditions (FBS/HS versus XF/SF; +/- MMC), as well as changes during adipogenic, osteogenic or chondrogenic inductions were monitored by light microscopy.
4.4.3 Viability of ASCs
In study IV, the cell attachment and viability were evaluated using quantitative Live/Dead staining method (Molecular Probes, Eugene, OR, USA). Briefly, samples were incubated for 45 min with a mixture of 5µM CellTrackerTM green (5- chloromethylfluorescein diacetate [CMFDA]; Molecular Probes) and 2.5µM Ethidium Homodimer-1 (EH-1; Molecular Probes). Fluorescence microscope was used to obtain cell images in which the viable cells stained green and red fluorescence indicated dead cells.
4.4.4 Proliferation assays
In studies I and II, the cell viability and metabolic activity in the different culture conditions (FBS, HS, and SF/XF) and in MMC condition were assessed with the PreMix WST-1 Cell Proliferation Assay System (study I) or Cell Counting Kit -8 (CCK-8) (study II) (Takara Bio Inc., Shiga, Japan). Both assays are based on the cleavage of tetrazolium salts (WST-1/WST-8) by mitochondrial dehydrogenase in viable cells, which enable to measure the cell proliferation and viability with colorimetric assay. Tetrazolium salts are cleaved to formazan dye by the succinate- tetrazolium reductase which exists in mitochondrial respiratory chain and is active only in viable and metabolically active cells. The amount of the formazan dye that is generated by the activities of dehydrogenases in cells is directly proportional to the number of living cells. The detection sensitivity of CCK-8 was higher than WST-1 tetrazolium salt. ASCs were seeded on 48-well plates at a density of 2,500 cells/cm2, and the proliferation was assessed at 1, 4, 7, and 11 days. In brief, at each time point, the cell-culture medium was removed, and DPBS (Dulbecco Phosphate-Buffered Saline, Lonza, BioWhittaker, Verviers, Belgium) and PreMix WST-1/CCK-8 reagent were added 10:1. The 48-well plate was incubated for 4 hours (I) or 3 hours (II) at 37°C, and the relative cell proliferation activity was measured in a microplate reader (Victor 1429 Multilabel Counter; Wallac; Turku, Finland) at 450 nm.
In study I, the population doubling was determined by using the formula x = log2(NH)/(N1), where N1 is the absorbance value at day 1, and NH is the absorbance value at observed time point 4, 7, or 11, as described previously (Cristofalo et al., 1998). To calculate the cumulative population doubling, the population doubling was determined in each passage and compared with the population doubling of earlier passages. In study II, the metabolic activity was
detected by CCK-8 Cell Proliferation Assay and was normalized to the number of cells in culture by quantifying the total DNA from of the cultures with CyQUANT®
cell proliferation assay.
In studies II and IV, the cell number was quantitatively analyzed by determining the amount of total DNA with a cell proliferation assay kit (CyQUANT®, Molecular Probes, Invitrogen, Paisley, UK). CyQUANT dye expresses fluorescence when bound to cellular nucleic acids. Briefly, cells were lysed using 0.1% Triton-X 100 buffer (Sigma-Aldrich), and the supernatant was collected and stored at -80°C until final analyses. Twenty micro liters of each sample were mixed with CyQUANT GR dye and lysis buffer in a 96-well plate (Nunc). Fluorescence signals were measured with a microplate reader at 480/520nm.