Supporting data of spatiotemporal proliferation of human stromal cells adjusts to nutrient availability and leads to stanniocalcin-1 expression in vitro and in vivo

This data article contains seven figures and two tables supporting the research article entitled: spatiotemporal proliferation of human stromal cells adjusts to nutrient availability and leads to stanniocalcin-1 expression in vitro and in vivo[1]. The data explain the culture of stromal cells in vitro in three culture systems: discs, scaffolds and scaffolds in a perfusion bioreactor system. Also, quantification of extracellular matrix components (ECM) in vitro and staining of ECM components in vivo can be found here. Finally the quantification of blood vessels dimensions from CD31 signals and representative histograms of stanniocalcin-1 fluorescent signals in negative controls and experimental conditions in vivo are presented.


Value of the data
Human stromal cell proliferation under various conditions. Multidisciplinary approach to understand and exploit the therapeutic potential of cells.
Results of implantation in mice of human cells in a novel well system.
1. Data, experimental design, materials and methods

Scaffold characterization
Microcomputed tomography (μCT, eXplore Locus SP μCT scanner, GE, Brussels, Belgium) at 14 μm resolution were used to characterize 2D and 3D scaffolds. Volume, porosity, and surface area of 2D and 3D scaffolds were determined with Microview software (Open source) as performed before [2]. Briefly, the threshold was adjusted to differentiate on the grayscale image between polymer voxels and pore voxels (One voxel was a 23 Â 23 Â 23 μm volume-element). The fraction of pore voxels within a scaffold determined its porosity. The pore size was determined by filling pore voxels with overlapping spheres [3]. The average size of a sphere occupying the pore voxel determined the average pore size. The boundaries between pore and polymer voxels determine the specific surface area. (Fig. 1 and Tables 1 and 2)

Perfusion bioreactor culture
A direct perfusion flow bioreactor configuration was used. The bioreactor was comprised of inner and outer housings made of polycarbonate (Applikon Biotechnology BV, Schiedam, The Netherlands), where PEOT/PBT cylindrical scaffolds (8 mm in diameter by 3 mm in height) were kept press-fit in the inner housing during cultivation. The bioreactor was connected to 3.2 mm PharMed tubing (Cole-palmer, The Netherlands), which was used throughout the system in a loop composed of: a supply vessel, 0.89 mm microbore tubing (Cole-palmer) used only on a pumphead (Masterflex, the Netherlands), fittings to connect 0.89-3.2 mm tubing, an oxygenator (explained below), the bioreactor, in-line oxygen and pH microsensors (Presens GmbH, Germany) and back to the supply vessel. One run was defined by four of these systems run in parallel for 8 days with medium refreshments twice a week, where each system contained an 8 Â 3 mm cylindrical scaffold dynamically seeded with 1.5 million cells and connected independently to their own oxygenator, tubing and αMEM proliferation medium supply.
To achieve flows in the range of 0.1-1 ml/min, a pump head (Masterflex, The Netherlands) was connected to the pump (Masterflex, The Netherlands), where 0.89 microbore tubing (Cole Palmer) was used. The four bioreactor systems were placed in a temperature-controlled box and kept at 37 1C, scaffolds (B). The radius of scaffolds is plotted against the surface area measurements (C), which allowed the production of 2D and 3D systems of PEOT/PBT with comparable surface areas.
providing per run four scaffolds (n¼4) with stromal cells for RNA extraction. These incubation units had to be supplied with a gas-controlled atmosphere. To supply the cells with oxygen and carbon dioxide, an oxygenator was built. The oxygenator comprised a closed chamber containing a gas-permeable silicon tube. The gas environment in the chamber was kept at a constant level of 21% O 2 and 5% CO 2 and medium was pumped through the gas-permeable tube at a flow rate of 0.3 ml/min. This system maintained the pH (7.1) at the bioreactor outlet during the culture period.
Chemo-optic flow-through micro oxygen sensors (FTC-PSt-3; Presens GmbH, Germany) that detect the quenching of luminescence by oxygen and an oxygen meter (Fibox-3; presens GmbH) were used as previously shown [4]. For 100% dissolved oxygen (DO) calibration, gas with the compositions mentioned above was supplied to the medium via the oxygenator. For 0% DO calibration, nitrogen gas was supplied through the medium via the oxygenator. Flow through cell (FTC-HP8-S, Presens GmbH) connected to pH-1 mini (Presens GmbH) were used to measure the pH of the medium at the outlet of the culture chamber.

Glycosaminoglycans (GAGs) and collagen quantification
GAGs were quantified on 2D discs and 3D scaffolds with 9-dimethylmethylene blue chloride (DMMB, Sigma-Aldrich) staining in PBE buffer: 14.2 g/l Na 2 HPO 4 and 3.72 g/l Na 2 EDTA, pH 6.5. A micro plate reader (Bio-TEK instruments) was used to spectrophotometrically determine absorbance at 520 nm. Chondroitin sulfate was used as standard.   Then, HPLC was performed as described before [5]. Collagen content was calculated from the total amount of hydroxyproline in each sample. It was assumed that there are 300 hydroxyproline residues per collagen molecule with a molecular weight of collagen of 300 kDa. (Fig. 3)