Methods Article Optimization of Culture Conditions for the Expansion of Umbilical Cord-Derived Mesenchymal Stem or Stromal Cell-Like Cells Using Xeno-Free Culture Conditions Tim Hatlapatka, M.Sc., 1 Pierre Moretti, Ph.D., 1 Antonina Lavrentieva, M.Sc., 1 Ralf Hass, Ph.D., 2 Nicole Marquardt, Dipl.Biol., 3 Roland Jacobs, Ph.D., 3 and Cornelia Kasper, Ph.D. 1 First isolated from bone marrow, mesenchymal stem or stromal cells (MSC) were shown to be present in several postnatal and extraembryonic tissues as well as in a large variety of fetal tissues (e.g., fatty tissue, dental pulp, placenta, umbilical cord blood, and tissue). In this study, an optimized protocol for the expansion of MSC-like cells from whole umbilical cord tissue under xeno-free culture conditions is proposed. Different fetal calf sera and human serum (HS) were compared with regard to cell proliferation and MSC marker stability in long-term expansion experiments, and HS was shown to support optimal growth conditions. Additionally, the optimal concentration of HS during the cultivation was determined. With regard to cell proliferative potential, apoptosis, colony-forming unit fibroblast frequency, and cell senescence, our findings suggest that an efficient expansion of the cells is carried out best in media supplemented with 10% HS. Under our given xeno-free culture conditions, MSC-like cells were found to display in vitro immunoprivileged and immunomodulatory properties, which were assessed by co-culture and transwell culture experiments with carboxyfluorescein diacetate succinimidyl ester- labeled peripheral blood mononuclear cells. These findings may be of great value for the establishment of biotechnological protocols for the delivery of sufficient cell numbers of high quality for regenerative medicine purposes. Introduction M esenchymal stem or stromal cells (MSC) are of great interest for cell-based therapies and tissue engineering approaches, as these cells are capable for extensive self- renewal and display a multilineage differentiation potential. 1–4 Additionally, MSC were shown to exhibit immunomodu- latory properties 5–8 and display supportive function through parakrine effects; that is, MSC support tissue repair by stimulating and modulating tissue-specific cells rather than differentiating into specialized cells. 9,10 Since first isolated in the mid-1960s, 11 bone marrow (BM)-derived MSC (BMSC) are currently almost exclusively used in clinical trials. Never- theless, the collecting procedure from BM is invasive and painful, and the number, differentiation potential, and maxi- mal life span of MSC from BM decline with increasing age. 12–14 Therefore, MSC isolated from alternative and easily accessible sources gained more and more attention over the last decade. MSC were found to be present in a variety of postnatal and extraembryonic tissues and organs as well as in a large variety of fetal tissues. 15–17 The tissue of the human umbilical cord (UC) has been found to be a rich source of MSC, 18–21 and because it is easily accessible, this tissue may represent a valuable alternative to BM as a source of MSC. Pioneer works of several groups during the last decade demonstrated that the tissues of the UC harbor MSC populations exhibiting potential for clinical applications (reviewed in Ref. 22 ). Low level of rejection was observed in transplantation studies in animals 23–26 and first reports strongly suggest that MSC derived from UC display similar immunoprivileged properties 27–29 as described for BMSC. Given these recent findings, UC-MSC may have a great potential for autologous as well as allogeneic trans- plantation to initiate tissue repair. The development of suitable biotechnological protocols for the ex vivo expansion of UC-MSC is a challenge to deliver a sufficient number of cells to a patient. MSC have been derived from UC tissue by various approaches using non- human serum (HS) such as fetal calf serum (FCS) for isola- tion and expansion of the cells. However, the use of sera of animal origin raises some safety concerns—in particular, the potential transmission of infections, for example, viruses and 1 Institute of Technical Chemistry, Leibniz University Hannover, Hannover, Germany. 2 Biochemistry and Tumor Biology Lab, Department of Obstetrics and Gynecology, Medical University, Hannover, Germany. 3 Department of Clinical Immunology and Rheumatology, Medical University, Hannover, Germany. TISSUE ENGINEERING: Part C Volume 17, Number 4, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tec.2010.0406 1