Serum‐Free Culture Conditions for Cells Capable of Producing Long‐Term Survival in Lethally Irradiated Mice

Abstract

The goal of ex vivo culture is to expand and/or differentiate cells in culture such that they retain their functional characteristics when reinfused into a patient. The studies presented here analyzed the use of culture conditions devoid of serum to expand murine hematopoietic stem cells. Bone marrow cells from male B6D2F1/J mice were cultured for up to 28 days in serum-free medium in the absence or presence of stem cell factor (SCF), GM-CSF or a combination of the two factors. Cells cultured for up to 21 days were assessed for granulocyte-macrophage colonyforming cells (GM-CFC), spleen colony-forming units, and cells responsible for short-term and long-term hematopoietic repopulation in lethally irradiated mice. Compared to initial seeding levels, the presence of SCF and GM-CSF increased total cell numbers 90-fold and GM-CFC numbers 42-fold over a 21-28 day culture period. Although spleen colony-forming unit cells did not increase, they were maintained at initial seeding levels over a 21-day period in the presence of SCF and GM-CSF. In lethally irradiated mice, survival enhancement and hematologic reconstitution were optimum with cells cultured for only seven days: survival at six months was 100% with cells cultured in SCF plus GM-CSF or SCF alone, compared to 50% with cells cultured with only GM-CSF. Hybridization analysis of bone marrow, spleen and thymus DNA from irradiated mice transplanted with these cultured cells confirmed male donor cell-derived repopulation at 45 days and 180 days post-transplant. These studies illustrate that murine GM-CFC can be expanded and that long-term repopulating hematopoietic cells can, at the minimum, be maintained ex vivo in serum-free culture. The use of defined serum-free culture systems holds great promise for further evaluation of the mechanisms that control hematopoietic stem cell proliferation.

Differential Kinetics of Primitive Hematopoietic Cells Assayed In Vitro and In Vivo During Serum‐Free Suspension Culture of CD34 + Blood Progenitor Cells

“…Whereas the former researchers all used serum‐supplemented media, Rebel and coworkers used serum‐free expansion of murine highly enriched progenitors and showed that, although the numbers of cells with a primitive (lineage‐negative wheat germ agglutinin‐positive) phenotype expanded, the engraftment potential of the cultured product was not amplified but could at best be maintained [17]. Similar results were also reported by Brown et al [39] after expansion of murine bone marrow cells in the presence of SCF and GM‐CSF. Recently, however, an increase in murine reconstituting cells could be demonstrated in a culture system starting from Sca‐1 + lin – bone marrow cells [40].…”

Maintenance and Self-Renewal of Long-Term Reconstituting Hematopoietic Stem Cells Supported by Amniotic Fluid

“…In the absence of AF, SCF alone did not support proliferation or differentiation of bone marrow cells, as has also been shown previously. Bone marrow cultures grown in the presence of SCF alone with (16) or without serum (44,45) does not promote the growth or self-renewal of hematopoietic stem cells.…”

A simple in vitro culture system for tracheal cartilage development

“…tr trachea, es esophagus, hr heart, V ventral, D dorsal. ences: (1) Our use of organ culture inserts instead of using Millipore filter and stainless steel grids allows organ explants to freely float and have greater access to culture media, (2) maintaining culture medium free of serum instead of their use of 25% fetal calf serum for culture media prevents problems associated with using serum such as antagonizing the growth and differentiation of cells in in vitro culture studies (Ambesi-Impiombato et al 1980;Brown et al 1997), and (3) supplementing culture media with transferrin to stimulate proliferation of mesenchymal cells (Ekblom et al 1983;Thesleff and Ekblom 1984) ensures production of sufficient amount of mesenchymal cells to grow cartilages in our culture studies. Despite the similarities, we believe that the differences that we implemented in our culture technique make our technique significant and effective in studying cartilage development.…”