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

^a Quality Biological, Inc., Gaithersburg, Maryland, USA;
^b The Armed Forces Radiobiology Research Institute, Bethesda, Maryland, USA

Key Words. Stem cell • Serum-free culture • Ex vivo expansion • Transplantation • Progenitor cells

Dr. Ronald L. Brown, Quality Biological, Inc., 7581 Lindbergh Drive, Gaithersburg, MD 20879, USA.

	Abstract

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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 colony-forming 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.

	Introduction

Top Abstract Introduction Materials and methods Results Discussion References

 
The ability to expand and/or differentiate cells in culture such that they retain specific functional characteristics when reinfused into patients holds numerous possibilities for the treatment of diseases [1, 2]. Ex vivo culture of hematopoietic cells has also been proposed to expand and differentiate myeloid cells for reinfusion into transplant patients as a means to overcome initial post-transplant neutropenia and susceptibility to opportunistic infections. Additional applications include a means to eliminate tumor cells from hematopoietic cells prior to reinfusing cells into patients and to support the transduction of specific genes into hematopoietic cells for gene therapy. The use of ex vivo culture as a means to expand hematopoietic progenitor/stem cells capable of reconstituting the hematopoietic system is most encouraging [3-15]. With these multiple proposed uses for ex vivo culture, it has become increasingly important to develop culture conditions that can be used to ascertain the full potential of this technology.

The ability of cells expanded in ex vivo culture to repopulate the bone marrow of lethally irradiated mice has been extensively evaluated [5-7, 9]. Such studies have attempted to determine the culture conditions necessary not only for the differentiation of hematopoietic stem cells, but also for the expansion of the long-term marrow repopulating cells. The culture conditions used to date have primarily been serum-containing basal media supplemented with various combinations of cytokines. One major disadvantage of using serum in media preparations is that it contains over 500 proteins, many of which are ill-defined and poorly characterized [16, 17], and some which have been shown to be antagonistic for growth and differentiation [18]. Furthermore, in the presence of some sera, cells have been reported to develop surface membrane associated determinates directed against serum components [19]. Thus, the requirement for serum to support the ex vivo expansion of hematopoietic stem/progenitor cells makes it difficult to ascertain and control the factors required to expand or differentiate the cells to specific lineages and may present immunological problems when the cells are reinfused.

In order to establish the usefulness of ex vivo culture for clinical regimens, it is necessary to develop culture conditions that are devoid of serum. As a step in this direction, we have evaluated the use of serum-free medium that, in the presence of appropriate cytokines, supports ex vivo expansion of murine hematopoietic stem/progenitor cells and, at a minimum, maintains long-term marrow repopulating cells.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion References

 
Mice
B6D2F1/J female and male mice (~20 g) were purchased from The Jackson Laboratories (Bar Harbor, ME). Mice were maintained in a facility accredited by the American Association for Accreditation of Laboratory Animal Care in Micro-Isolator cages on hardwood-chip, contact bedding and were provided commercial rodent chow and acidified water (pH 2.5) ad libitum. Animal rooms were equipped with full-spectrum light from 0600 to 1800 h and were maintained at 21°C ± 1°C and 50% ± 10% relative humidity with at least 10 air changes per hour of 100% conditioned fresh air. Upon arrival, all mice were tested for Pseudomonas and quarantined until test results were obtained. Only healthy mice were released for experimentation. All animal experiments were approved by The Armed Forces Radiobiology Research Institute (AFRRI; Bethesda, MD) Animal Care and Use Committee prior to performance.

Irradiation
The ⁶⁰Co source at AFRRI was used to administer bilateral total-body ⁶⁰Co gamma radiation. Mice were placed in ventilated Plexiglas containers and irradiated at a dose rate of 0.4 Gy/min. Dosimetry was performed using ionization chambers with calibration factors traceable to the National Institute of Standards and Technology. Before experiments were initiated, the dose rate at the midline of an acrylic mouse phantom was measured with a 0.5 cm³ tissue-equivalent ionization chamber manufactured by Exradin (Lisle, IL). Before each experimental irradiation, the dose rate at the same location with the phantom removed was measured with a 50 cm³ ionization chamber fabricated at AFRRI. The ratio of these two dose rates, the tissue-air ratio, was then used to ensure delivery of the midline dose desired for each animal exposure. The tissue-air ratio in these experiments was 1.00.

Bone Marrow
The femurs and tibias were removed from male mice after cervical dislocation. The bone marrow was flushed from the bones with Iscove's modified Dulbecco's medium (IMDM; Quality Biological, Inc.; Gaithersburg, MD) and single cell suspensions were prepared by repeatedly drawing the medium and cells through a 25 gauge needle.

Liquid Cell Culture
Bone marrow cells were cultured in QBSF-58 (Quality Biological, Inc.), a serum-free medium especially designed for hematopoietic cell growth. QBSF-58 contains IMDM basal medium plus bovine serum albumin, human transferrin, a water soluble source of cholesterol, and bovine insulin. For those experiments using serum-containing medium the basal medium was IMDM plus 20% FBS (fetal bovine serum; Quality Biological, Inc.). Prior to use, 2 mM of L-glutamine, 50 U/ml of penicillin and 50 µg/ml of streptomycin (Quality Biological, Inc.) were added to each medium. The cells were cultured in the absence or presence of 125 ng/ml of recombinant murine stem cell factor (rmSCF; with carrier proteins; R & D Systems; Minneapolis, MN), 10 ng/ml of rmGM-CSF (with carrier proteins; R & D Systems), or a combination of both cytokines. Stock solution of these cytokines was diluted in QBSF^®58 medium. The cells were cultured in 24 mm porous cell culture inserts (Corning; Cambridge, MA) at a density of 1.5 x 10⁶ cells in 1.5 ml of medium per insert. The inserts were then placed inside six-well culture plates containing 2.6 ml of medium per well. Depleted medium-containing cytokines were replaced with fresh medium and cytokines at seven day intervals. Upon reaching a density of greater than 3 x 10⁶ cells/well, the cells were subcultured to 1.5 x 10⁶ cells/well. The cultures were incubated at 37°C in a fully humidified atmosphere of 5% CO₂ and air. An aliquot was removed from each culture well on the days indicated and the number of viable cells ascertained using the trypan blue dye exclusion assay. To ensure that cells were not adhering to the transwell inserts, on days 7, 14, 21 and 28 the inserts were trypsinized for 5-10 min then counted. No increase in cell numbers was observed after trypsinization indicating that the cells were not adhering to the inserts. All cell counts are expressed as the number of viable cells per ml in the inserts and have been adjusted for cell loss during medium removal for counting.

Granulocyte-Macrophage Colony-Forming Cell (GM-CFC) Assay
GM-CFCs were grown on a cytokine-supplemented soft-agar medium in 35 mm culture dishes (One Step Bone Marrow Kit, Quality Biological, Inc.) [20]. The soft-agar matrix was comprised of methylcellulose (Dow Chemical Co.; Midland, MI) and Seaplaque (FMC Bioproducts; Rockland, ME) in IMDM supplemented with 30% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml; all from Quality Biological, Inc.) and rmGM-CSF (40 ng/ml; R & D Systems). The cultures were incubated at 37°C in a fully humidified atmosphere of 5% CO₂ and air for 7-9 days. Only those colonies consisting of greater than 50 cells were scored.

Spleen Colony-Forming Unit (CFU-S) Assay
CFU-S were evaluated by the method of Till and McCulloch [21]. Recipient mice were exposed to 9 Gy of total-body ⁶⁰Co radiation to eradicate endogenous hemopoietic stem cells. Three to five h later, bone marrow cells (5 x 10⁴) were injected i.v., via the lateral tail vein, into the irradiated recipients. Twelve days after transplantation, the recipients were euthanized by cervical dislocation and their spleens were removed. The spleens were fixed in Bouin's solution, and the grossly visible spleen colonies were counted. Each treatment group consisted of at least seven mice.

Survival Studies
Cultured or fresh male bone marrow cells (5 x 10⁴) were injected i.v., via the lateral tail vein, into female mice that had been irradiated 3-5 h previously with 9 Gy total-body ⁶⁰Co. The mice were then monitored daily for survival. At 45 days post-transplant, half of the survivors were euthanized to determine the percentage of the bone marrow, spleen and thymus cells that had repopulated from the transplanted male donor cells. At 180 days post-transplant, the remaining mice were euthanized and the percentage of the bone marrow, spleen and thymus cells that had repopulated from the transplanted donor cells was similarly ascertained.

Determination of Donor Cell Engraftment
At euthanasia, each animal was individually identified and genomic DNA was extracted from the bone marrow (femurs and tibias), spleen and thymus. Immediately following excision, spleen and thymus tissue was washed with cold Dulbecco's phosphate-buffered saline (D-PBS) and cut into 50-100 mg pieces. The tissue pieces were quick-frozen on dry ice and stored at -80°C until used for DNA extraction. After pulverizing the frozen tissue with a mortar and pestle, genomic DNA was extracted using Proteinase K and anionic exchange matrix isolation columns (A.S.A.P. Genomic DNA Isolation Kit, Boehringer Mannheim; Indianapolis, IN). Bone marrow was aspirated from mouse bones in D-PBS using a 25 gauge hypodermic needle. The cells were washed with cold D-PBS by centrifugation and genomic DNA was extracted using the A.S.A.P. Genomic DNA Isolation Kit. Concentration and purity of the resultant DNA was determined spectrophotometrically.

Total genomic DNA from each tissue (2 µg) was blotted in a dot format onto a Zeta-Probe nylon membrane (Bio-Rad Laboratories; Hercules, CA), incubated overnight with digoxigenin-labeled probes at 68°C, and the hybridized product detected with the chemiluminescent substrate CSPD (Genius Nonradioactive Nucleic Acid Labeling and Detection System, Boehringer Mannheim). Exposed films were evaluated by densitometry (Bio-Rad Model 620 Video Densitometer and 1-D Analyst II software). Membranes were stripped for rehybridization using 0.2 N sodium hydroxide (NaOH) and 0.1% sodium dodecyl sulfate. Male-female spleen or thymus DNA mixtures of 1%, 10%, 25%, 50% and 100% male DNA dotted on the right and left side of each membrane were used as controls for all hybridization reactions. Murine Y chromosomal sequences were detected with a 1.5 kb DNA fragment derived from Eco RI digestion of plasmid 145C5 [22] that was digoxigenin-labeled by random priming. DNA mixtures containing 10% (200 ng) male DNA were reproducibly quantitated by densitometry in these hybridization reactions (Fig. 1). All optical density area values of less than 1.0 (equivalent to approximately 10% male DNA) were considered to be negative in this analysis. The area calculated from the densitometry scan was proportional to the amount of male DNA comprising the sample in the range of 25% (500 ng) to 100% (2 µg).

View larger version (13K): [in this window] [in a new window]   Figure 1. Relationship of area of optical density scan to percentage of male DNA in 2 µg DNA dot blot in 45 day and 180 day analysis. Mean ± standard error.

Statistical Analysis
Results are expressed as the mean ± SE. Groups were compared by ANOVA analysis performed using the Graph Pad Instat software.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Cellular Proliferation
The ability of bone marrow cells to proliferate in serum-free medium with and without the addition of cytokines was evaluated by removing aliquots from the cultures at the noted times and performing total and viable cell counts. In eight separate experiments, the ability of the bone marrow cells to proliferate in serum-free medium supplemented with SCF and/or GM-CSF was evaluated (Fig. 2). In the presence of both SCF and GM-CSF, cellularity increased approximately 90-fold over the initial seeding level by day 28. In four of the above experiments, bone marrow cells were also cultured in serum-free medium in the presence of either SCF alone or GM-CSF alone (Fig. 1). In the presence of only SCF, viable cell numbers increased from the initial seeding level of 1 x 10⁶/ml to 2.9 ± 0.4 x 10⁶ cells/ml at day 7, however, this was followed by a decline in cell number to 1.6 ± 0.2 x 10⁶ cells/ml by day 21. Cells cultured in the presence of only GM-CSF also increased slightly from the initial seeding level of 1 x 10⁶/ml to 2.1 ± 0.4 x 10⁶/ml by day 14, but declined to 1.8 ± 0.4 x 10⁶ cells/ml by day 21. In the absence of any added cytokines, viable cell numbers decreased from an initial seeding level of 1 x 10⁶/ml to 4.8 ± 0.03 x 10⁵ cells/ml by day 10 and remained near this level through day 28. Thus, maximum cellular proliferation occurred in the presence of a combination of SCF and GM-CSF.

View larger version (17K): [in this window] [in a new window]   Figure 2. Proliferation of murine bone marrow cells cultured under serum-free conditions in the presence of SCF alone (125 ng/ml), GM-CSF alone (10 ng/ml) or a combination of both cytokines. Mean ± the standard error.

Expansion of GM-CFC Progenitor Cells
To assess the ability of GM-CFC to proliferate and expand in serum-free cultures, aliquots of the cultured bone marrow cells were removed and assessed for GM-CFC in the presence of GM-CSF (Fig. 3). In serum-free medium without any cytokines, GM-CFC numbers rapidly declined from an average initial seeding level of 2,406 ± 143/ml to only 44 ± 12/ml by day 7; by day 10 almost no GM-CFC could be detected. In the serum-free cultures containing both SCF and GM-CSF, the GM-CFC numbers increased from the initial average seeding level of 2,406 ± 143/ml to 100,000 ± 18,000/ml by day 17, representing an approximate 42-fold increase over the initial seeding level. In four of the above eight experiments, GM-CFC expansion was also evaluated in cells cultured in serum-free medium in the presence of only SCF or only GM-CSF (Fig. 3). The presence of SCF alone maintained GM-CFC at levels nearly equivalent to the initial seeding level of 2,406 ± 143/ml for 14 days, however, GM-CFC declined to 1,466 ± 307/ml by day 21. In the presence of GM-CSF alone, GM-CFC levels declined from the initial seeding level of 2,406 ± 143/ml to 235 ± 10/ml by day 7 and remained near this level through day 21. Thus, the presence of both SCF and GM-CSF was necessary to expand GM-CFC in the serum-free cultures.

View larger version (19K): [in this window] [in a new window]   Figure 3. Expansion of murine bone marrow GM-CFC in serum-free medium supplemented with SCF alone (125 ng/ml), GM-CSF alone (10 ng/ml), or a combination of both cytokines. Mean ± the standard error.

Maintenance of CFU-S
The ability to expand CFU-S in cytokine-supplemented serum-free cultures was evaluated in three separate experiments. In the absence of any cytokines, CFU-S levels rapidly declined from an initial seeding level of 560 ± 25/ml to barely detectable levels by day 14. In the presence of both SCF and GM-CSF, CFU-S were maintained at levels equivalent to the initial seeding level for up to 21 days. In cultures containing GM-CSF or SCF, there was a rapid decline of CFU-S to 32 ± 12 CFU-S/ml and 74 ± 3 CFU-S/ml, respectively, at day 7. Thus, the presence of both SCF and GM-CSF was necessary to maintain CFU-S in the serum-free medium (Fig. 4).

View larger version (17K): [in this window] [in a new window]   Figure 4. Maintenance of murine bone marrow CFU-S in serum-free medium supplemented with SCF alone (125 ng/ml), GM-CSF alone (10 ng/ml), or a combination of both cytokines. Mean ± the standard error.

View larger version (17K): [in this window] [in a new window]   Figure 5. Survival of irradiated female mice transplanted with syngeneic male bone marrow cells cultured in serum-free medium supplemented with SCF alone (125 ng/ml), GM-CSF alone (10 ng/ml), or a combination of both cytokines. Ten mice per group were exposed to 9 Gy 60Co, transplanted with 5 x 104 cells, and survival was monitored daily for 45 days. It should be noted that no additional animals died over days 45 to 180.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

It is important to note that the serum-free culture conditions noted herein did not support the attachment of the traditional adherent cell layers on plastic tissue culture dishes as noted for serum-containing medium. Coating the surfaces of the tissue culture plates with collagen, ploy-D-lysine, or fibronectin also did not facilitate attachment under serum-free culture conditions. Thus, the serum-free culture conditions also eliminate the interaction between an adherent cellular layer and nonadherent cells.

In the present study, unfractionated murine bone marrow cells expanded nearly 90-fold when cultured for 14-21 days in serum-free medium plus SCF and GM-CSF; furthermore, the number of GM-CFC expanded 42-fold over the initial seeding level. Neither SCF or GM-CSF alone significantly increased numbers of the total cells or GM-CFC. This data is consistent with previous studies using short-term (6-7 days) ex vivo culture of murine cells in serum-containing media [8, 9] and, therefore, supports the feasibility of using serum-free media in such studies. Due to the rapid ex vivo expansion of the total cell number in the presence of only SCF and GM-CSF, we decided to evaluate which cell populations were being modulated in culture.

In contrast to GM-CFC, CFU-S numbers did not expand in our culture system. However, when media was supplemented with SCF and GM-CSF, CFU-S numbers were maintained at the initial seeding level for up to 21 days. In contrast, in the presence of SCF alone or GM-CSF alone, CFU-S numbers dropped below the initial seeding level. These effects contrast with results of studies using serum-containing medium where CFU-S numbers were reported to increase 10-fold in the presence of SCF alone [8]. In the former studies, CFU-S expansion may have been due to synergy between the SCF and unknown cytokines present in the serum. Despite this difference, our data indicates that CFU-S can, at the minimum, be maintained in serum-free cultures which simultaneously expand GM-CFC progenitors.

In the present study, we also evaluated the ability of serum-free cultured murine bone marrow cells to engraft into lethally irradiated recipient mice. Our data indicates that bone marrow cells cultured in the presence of SCF for up to 14 days or in the presence of SCF and GM-CSF for seven days could rescue 90%-100% of the irradiated mice. Cells cultured for longer times, or cells cultured in GM-CSF alone, resulted in fewer numbers of survivors. This decrease in the number of survivors in the presence of GM-CSF alone or SCF and GM-CSF after seven days may be due to the GM-CSF differentiating the cells to the myeloid lineage and short-term marrow repopulating compartment rather than maintaining them in the long-term marrow repopulating compartment. This survival data is consistent with that of others who have used bone marrow cultured for 6-7 days in the presence of FBS and various cytokine combinations [8, 9]. The presence of donor cells in the bone marrow, spleen and thymus of the surviving mice at 45 days and 180 days post-transplantation was also verified. This data suggested that serum-free culture conditions can support both short-term and long-term marrow repopulating cells.

Recently, ex vivo expanded CD34⁺ cells supplemented with autologous progenitors have been transplanted into patients undergoing high-dose chemotherapy for solid tumors [23, 24]. These studies illustrated the safety and feasibility of transplanting ex vivo expanded cells; however, neither study yielded better results than obtained with autologous transplantation of unseparated bone marrow mononuclear cells or positively selected CD34⁺ cells. Further studies need to be performed to evaluate serum-free medium comprised of pasteurized human proteins rather than QBSF-58 medium containing bovine protein, as well as the culture conditions, cytokines and cell types that will direct cellular proliferation and/or differentiation to the desired results. The use of serum-free medium in these studies represents an important alternative to the use of serum and provides a basis for further studies with human hematopoietic stem/progenitor cells. Such studies will be valuable in determining the success of ex vivo culture as a treatment modality.

	Acknowledgments

 
This work was supported by NIH grant 2 R44 DK46223-02.

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