Introduction

Mesenchymal stem cells (MSCs) are under active investigation in tissue engineering and cell therapy research. Traditionally, MSCs were isolated from bone marrow (BM) and cultured in medium containing fetal bovine serum. This poses particular challenges to the use of human MSCs for therapeutic applications, as serumbased culture methods introduce contamination and variability concerns, MSCs occur at low frequency in bone marrow, and bone marrow donation is a slow and painful process.

The ability to derive MSCs from more practical sources and culture them in serum-free environments would therefore address several stumbling blocks in translational MSC research. MSCs have previously been isolated from adipose tissue (Zuk et al., 2001), which is abundant and relatively easy to procure. Adipose tissue would thus be a more practical source for harvesting MSCs.

This technical bulletin presents protocols and data on a serum and xeno-free medium (MesenCult™-XF) for the ex vivo expansion of MSCs isolated from adipose tissue (Figure 1). The data indicate that adipose tissue-derived MSCs (AT-MSCs) expand much more extensively in MesenCult™-XF as compared to a traditional serum-based medium (Figure 2), while maintaining MSC cell surface phenotypes (Figure 3) and multi-lineage differentiation potential (Figure 4). Culturing AT-MSCs with serum-free, xeno-free MesenCult™-XF Medium provides a convenient method of obtaining MSCs for translational research.

Results

AT-MSC Protocols

Sample Preparation

Adipose tissue (AT) samples were obtained from reduction mammoplasty and prepared for culture using the protocol below:

1. Finely mince tissue with a scalpel.
2. Digest minced tissue in 5 mL of 0.25% Collagenase Type I per cm³ of tissue for 60 minutes at 37°C in a shaking water bath.
3. Following digestion, place the samples upright for 5 minutes, to allow separation of the lipid layer from the aqueous layer.
4. Remove the lipid and adipose cells with a pipette or aspirator.
5. Wash the remaining cells by bringing the total volume in the tube to 50 mL with Buffer (PBS with 2% FBS + 1 mM EDTA).
6. Filter the cells through a 70 μm cell strainer to remove cell aggregates and connective tissue debris.
7. Centrifuge the cells at 400 x g (1200 rpm) for 10 minutes.
8. Remove the supernatant and resuspend the cells in 1 - 2 mL Buffer.
9. Dilute cells 1/50 in 3% Acetic Acid with Methylene Blue and count the total number of nucleated cells using a hemacytometer.

Cell Culture

MSCs were cultured either in serum-free, xeno-free medium (MesenCult™-XF) or in a traditional serum-containing medium (Control), using the protocol below:

1. Coating: For cells cultured in MesenCult™-XF, 6-well culture plates were first coated with MesenCult™-SF Attachment Substrate (required for cell adherence under serum-free culture conditions). Uncoated tissue culture treated plates were used for the Control cultures.
2. Plating: Primary cells were plated at 0.5 - 1.0 x 10³ cells/cm² in MesenCult™-XF, and at 2.5 - 5 x 10³ cells/cm² in Control medium, in a volume of 2 mL per well. It is important to plate a range of cell densities when starting with primary tissue, as variability is inherent within each sample.
3. Passaging: At each passage, MesenCult™-XF cultures were dissociated with MesenCult™-ACF Dissociation Kit, while Control cultures were dissociated with Trypsin-EDTA.
4. Subculturing: Following dissociation, cultured AT-MSCs were re-plated at 1.5 - 4 x 10³ cells/cm² in MesenCult™-XF, and at 0.5 - 1 x 10⁴ cells/cm² in Control medium.

Cell Assays

1. Expansion: MSCs were cultured as described above. To determine the number of cells at each passage, viable cells were counted by taking an aliquot of the harvested single cell suspension and using the Trypan Blue exclusion assay on a hemacytometer.
2. Cell Surface Phenotype: Passage 2 AT-MSCs cultured in MesenCult™-XF or Control medium were harvested, washed and labeled with fluorescent-conjugated (PE, FITC or APC) antibodies to CD105, CD90, CD73, CD45, CD11b and CD34. Flow cytometry analysis was performed using the BD FACSCalibur™.
3. Differentiation: Passage 2 AT-MSCs cultured in MesenCult™-XF were differentiated to the osteogenic lineage using the MesenCult™ Osteogenic Stimulatory Kit (for MSCs cultured in MesenCult™-XF), and to the adipogenic lineage using the MesenCult™ Adipogenic Stimulatory Supplement (Human). The differentiation procedures are detailed in the Technical Manual: Culture of Human Mesenchymal Stem Cells Using MesenCult™-XF Medium (Manual Catalog #29184).

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