Serum‐free media for cultures of primitive and mature hematopoietic cells

Sandstrom, Craig E.; Miller, William M.; Papoutsakis, Eleftherios T.

doi:10.1002/bit.260430806

Cited by 39 publications

(13 citation statements)

References 175 publications

(11 reference statements)

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“…Therefore, it is possible that the amount of iron and transferrin in the culture medium was insufficient for adequate heme synthesis, which resulted in lower intracellar heme content in highly proliferating cells under EPO stimulation. Actually, iron-saturated transferrin is essential in erythroid cell cultures when serum-free media are used (Sandstrom et al, 1994). However, in the present study, the culture medium was supplemented with FBS mostly containing iron and transferrin.…”

Section: Discussionmentioning

confidence: 89%

A simple method for enrichment of polychromatic erythroblasts from rat bone marrow, and their proliferation and maturation in vitro

et al. 2011

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-To evaluate the effects of a variety of chemical, biological and physiological stimuli on erythropoiesis, in vitro assays using erythroid progenitor cells from humans or laboratory animals are well-known methods. On the other hand, little has been reported on in vitro assays using mature erythroblasts such as polychromatic erythroblasts. In the present study, we established a convenient method for enrichment of polychromatic erythroblasts from rat bone marrow and confirmed their development in vitro. To establish a method for the enrichment of polychromatic erythroblasts, bone marrow cells from 3-and 10-week-old rats were separated by discontinuous density gradient centrifugation using Percoll. As a result, polychromatic erythroblasts were most highly enriched in the bone marrow fraction from 3-week old rats at the density interface between 1.040 and 1.058 g/ml. The enriched polychromatic erythroblasts were then cultured in growth medium supplemented with 20% fetal bovine serum in the presence or absence of erythropoietin for 48 hr. During the culture period, cell proliferation and maturation to orthochromatic erythroblasts were observed, and intracellular heme contents were also increased. In particular, the culture in the presence of erythropoietin revealed higher proliferation of erythroid cells, and therefore might be more appropriate for in vitro experiments on the effects of various stimuli on late-stage erythropoiesis.

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Section: Discussionmentioning

confidence: 89%

A simple method for enrichment of polychromatic erythroblasts from rat bone marrow, and their proliferation and maturation in vitro

et al. 2011

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“…Serum-free media have been analyzed and compared for their performance in ex vivo expansion of human hematopoietic progenitors (reviewed in Sandstrom et al, 1994). A recent study systematically compared the performance of various serum-free media (referred to as whole-serumdeprived media) in cord blood cell and BPC expansion, using SCF, IL-3, and IL-6 as growth factors (Sandstrom et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Serum-free ex vivo expansion of CD34+ hematopoietic progenitor cells

Möbest

Mertelsmann

Henschler

1998

Biotechnol. Bioeng.

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In an effort to obtain defined culture conditions for ex vivo expansion of hematopoietic stem and progenitor cells which avoid the supplementation of serum, we cultured human CD34+ hematopoietic progenitor cells in a chemically defined, serum‐free medium in the presence of hematopoietic growth factors (HGFs), stem cell factor (SCF), interleukin (IL)‐1β, IL‐3, IL‐6, and erythropoietin (EPO). A medium, SFM‐1, was prepared according to a protocol previously optimized for semisolid progenitor cell assays containing Iscove's Modified Dulbecco's Medium (IMDM) plus cholesterol, bovine serum albumin, transferrin, nucleotides and nucleosides, insulin, and β‐mercaptoethanol. In static cultures seeded with CD34+‐enriched progenitor cells isolated from human peripheral blood, a mean 76.6‐fold expansion of total nucleated cells and a mean 4.6‐fold expansion of colony‐forming cells (CFC) was recorded after 14 days. Morphological analysis of the expanded cells revealed formation of myeloid, erythroid, and megakaryocytic cells. Flow cytometric analysis indicated that CD34+ antigen expressing cells were maintained to a limited degree only, and cell populations expressing surface markers for myeloid (CD33, CD14, and CD15) and megakaryocytic (CD41a) lineages predominated. Within SFM‐1, bovine serum albumin (BSA), cholesterin, and transferrin represented the most critical components needed for efficient total cell and CFC expansion. Addition of autologous patient plasma (APP) or fetal calf serum (FCS) to SFM‐1 resulted in inferior cell amplification and CFC formation compared to controls in SFM‐1, indicating that the components used in SFM‐1 could replace exogenous serum. Four commercially available serum‐free media resulted in either comparable or lower total cell and CFC yields as SFM‐1. The transplantation potential of CD34+ cells after culture in SFM‐1 was assayed using limiting dilution analysis on preformed irradiated bone marrow stroma and revealed maintenance of long‐term bone marrow culture initiating cell (LTCIC) levels during the culture period. These data indicate that HGF‐supported multilineage ex vivo expansion of human CD34+ hematopoietic progenitor cells is feasible using an IMDM‐based culture medium which contains a restricted number of additives, resulting in analogous or improved yields of both primitive and differentiated cells compared to previously established protocols. We suggest that this culture protocol is of advantage when working with pharmaceutical‐grade preparations under serum‐free conditions. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 341–347, 1998.

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“…Unfortunately, the development of alternatives to animal sera for accessory cell-containing cultures has not been as successful (21)(22)(23), probably in part because of the significantly lesser effort expended on this application as compared with CD34-enriched cell expansion. This study, therefore, evaluated several alternatives to animal sera for the support of perfused BM MNC cultures, with the goal of using such an alternative in ongoing human clinical trials.…”

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confidence: 97%

Alternatives to Animal Sera for Human Bone Marrow Cell Expansion: Human Serum and Serum-Free Media

Koller

Maher²,

Manchel³

et al. 1998

Journal of Hematotherapy

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The increasing use of cultured human cells in clinical trials is highlighting the need for alternatives to media containing animal sera that are typically used to support these cultures. Perfused cultures of BM mononuclear cells (MNC) were used to evaluate animal sera alternatives with respect to the output of primitive, progenitor, and stromal cells. A serum level of 20% was optimal, and this could be provided by FBS alone or by a mixture of horse serum (HoS) and FBS, but not by HoS alone. Allogeneic human plasma (20%) supported half the level of cell, CFU-GM, and LTC-IC output as compared with animal sera-containing control. Significant donor-to-donor variability in human plasma was observed, but this was mitigated by pooling of plasma samples. Autologous and allogeneic human plasma performed equivalently. The use of autologous or allogeneic human serum was found to be equivalent to the use of human plasma, but all were inferior to animal sera. Animal sera supported typical stroma and cobblestone formation, whereas stroma in human serum cultures was less dense. Eight commercial serum-free media were tested and found to support MNC expansion to varying degrees, but none approached the performance of the animal serum-containing control, particularly with respect to stromal (i.e., CFU-F) support. In fact, when MNC were cultured in parallel with CD34-enriched cells, output (from MNC) was higher only in control medium, apparently because serum-free media reduced accessory cell effects. Because of these results, a new serum-free medium was developed for MNC cultures. This formulation outperformed all commercial serum-free media, resulting in cell and LTC-IC output equivalent to that of control. However, CFU-GM and CFU-F output were 66% and 9% of control, respectively. Precoating the culture surface with collagen increased CFU-F (and Thy-1+ cell) output to control levels, although CFU-GM output was still lower than control. The addition of either fibronectin or PDGF had no measurable effect, nor did the use of 5-100-fold greater concentrations of growth factor supplementation. The serum-free medium also increased CD41+ and CD61+ cell output to 150%-220% of control levels. The development of this new serum-free medium has potential for use in the perfused BM MNC culture systems currently in clinical trials to test the efficacy of expanded cells after cytoablative chemotherapy.

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Serum‐free media for cultures of primitive and mature hematopoietic cells

Cited by 39 publications

References 175 publications

A simple method for enrichment of polychromatic erythroblasts from rat bone marrow, and their proliferation and maturation in vitro

A simple method for enrichment of polychromatic erythroblasts from rat bone marrow, and their proliferation and maturation in vitro

Serum-free ex vivo expansion of CD34+ hematopoietic progenitor cells

Alternatives to Animal Sera for Human Bone Marrow Cell Expansion: Human Serum and Serum-Free Media

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