FBS free culture of porcine umbilical cord matrix cells

Abstract

The common choice of medium for culturing pig umbilical cord matrix stem cells (PUCs) is high glucose Dulbecco’s Minimum Essential Medium (HG-DMEM) supplemented with fetal bovine serum (FBS). FBS is a chemically undefined supplement that encourages attachment of explants and cells and is useful for long-term proliferation in an undifferentiated state. Removing FBS from the culture medium would decrease the possibility of microbial contamination and might produce more consistent results. A defined medium would facilitate experiments to determine requirements for specific growth factors and nutrients. Starting PUCs in a FBS-free environment proved to be a challenge. The results of 15 experiments testing various media, supplements, and culture conditions indicate that PUCs initially plated in an FBS-free environment do not attach as readily as those in HG-DMEM supplemented with FBS. PUCs were collected using enzyme digestion of the whole cord or by plating explants from the cord in culture medium. In the final experiment PUCs were seeded in 24-well plates (5.0 * 10[superscript]4 viable cells per well) with a collagen coating and cultured in Knock-out DMEM (KO-DMEM) with basic fibroblast growth factor (5ng/mL) and platelet derived growth factor (5ng/mL) in a low oxygen atmosphere (5% O[subscript]2/ 5% CO[subscript]2/ 90% N[subscript]2). The total non-adherent cell count at passage 1 was 1.78 * 10[superscript]5 +or- 3.68 * 10[superscript]4 and the total adherent cells were 2.58 * 10[superscript]5 +or- 9.29 * 10[superscript]4. The well confluence during initial cell proliferation appeared similar to cells cultured in the control media with 20% FBS (total adherent cells = 6.40 * 10[superscript]5 +or- S.E. 1.61 * 10[superscript]5 and total non-adherent cells = 2.88 * 10[superscript]5 + 7.60 * 10[superscript]4). However the number of adherent cells recovered for passage 2 was considerably less for cultures in FBS-free media than for the control group. Serum may affect attachment by providing attachment factors or it could change expression of integrins or other attachment molecules on the PUCs that enhance attachment to plastic or other substrates. In future studies the requirements for attachment of PUCs should be further evaluated.