An improved procedure for the development of human mast cells from dispersed fetal liver cells in serum-free culture medium

Journal of Immunological Methods 240 (2000) 101–110 www.elsevier.nl / locate / jim

An improved procedure for the development of human mast cells from dispersed fetal liver cells in serum-free culture medium Naotomo Kambe a , Michiyo Kambe a , Hyeun-Wook Chang a , Atsushi Matsui a , Hae-Ki Min a , Mousa Hussein a , Carole A. Oskerizian a , Jarko Kochan b , Anne-Marie A. Irani a , Lawrence B. Schwartz a , * a

Department of Internal Medicine, and Paediatrics, Medical College of Virginia, Virginia Commonwealth University, P.O. Box 980263, Richmond, VA 23298 -0263, USA b Hoffmann-La Roche, Nutley, NJ, USA Received 3 February 2000; accepted 1 March 2000

Abstract The in vitro development of human mast cells from fetal liver cells with recombinant human stem cell factor in serum-containing RPMI was compared to that in AIM-V media with and without serum. Compared to serum-containing media, AIM-V medium caused mast cells to develop earlier and in greater numbers. By 2 weeks, about 60% of cells in serum-free AIM-V medium were phenotypic mast cells, |2 times the percentages in serum-containing media. By 6 weeks the percentages of mast cells were $80% under all conditions, but the number of mast cells was 3–4-fold greater in serum-free AIM-V medium than in serum-supplemented media. Mast cells obtained in serum-free AIM-V medium exhibited rounded nuclei, like tissue-derived mast cells; mast cells obtained in serum-supplemented media had segmented nuclei. By 10–12 weeks of culture about 40% of the AIM-V-derived cells showed strong chymase immunocytochemical staining, a pattern observed for only 14% of the cells in serum-containing media. AIM-V medium is a suitable medium for the development of human mast cells in vitro, and permits an earlier, more selective and greater expansion of mast cells than serum-containing media.  2000 Elsevier Science B.V. All rights reserved. Keywords: Human mast cell; Serum-free culture medium; Tryptase; Chymase; Kit

1. Introduction Abbreviations: AEC, 3-amino-9-ethylcarbazole; AP, alkaline phosphatase; CPSR, controlled process serum replacement; DMEM, Dulbecco’s modified Eagle medium; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; mAb, monoclonal antibody; PBS, phosphate-buffered saline; rhSCF, recombinant human stem cell factor; TTBS, Tris-buffered saline containing Tween-20 *Corresponding author. E-mail address: [email protected] (L.B. Schwartz)

Mast cells play a central role in the pathophysiology of allergic disorders. Two types of human mast cells have been identified based on their composition of neutral proteases (Irani et al., 1986; Irani, 1995). MC TC cells contain tryptase, chymase, mast cell carboxypeptidase and cathepsin G in their secretory granules, and are the predominant type of mast cell

0022-1759 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0022-1759( 00 )00174-5

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in skin and in intestinal submucosa. MC T cells also contain tryptase in their granules, but without these other proteases, and are the predominant type of mast cell in alveolar wall and in small intestinal mucosa. Studies of human mast cells have been impeded by their restricted availability both from tissues and from in vitro culture systems. Recombinant human stem cell factor (rhSCF)-dependent development of human mast cells in vitro has been achieved with hematopoietic progenitor cells obtained from bone marrow and peripheral blood (Valent et al., 1992; Rottem et al., 1994), cord blood (Mitsui et al., 1993; Saito et al., 1996) and fetal liver (Irani et al., 1992a), but only after 3–4 weeks can most of the cells be identified as mast cells, and numbers of mast cells typically do not exceed the number of cells initially placed into culture. The addition of IL-6 to cultures of cord blood cells enhances proliferation of mast cells in some cases (Saito et al., 1996), but not in others (Oskeritzian et al., 1999), and has had no apparent effect on mast cell development or survival in the fetal liver cell system (Schwartz et al., unpublished results). Most mast cell culture systems utilize serum-containing media. Serum may provide hormones, mitogenic peptides and growth factors, vitamins, metal iron transport proteins, and other agents that are beneficial to mast cell development. Serum also contains agents that may adversely affect mast cell development. The present study shows that serumfree AIM-V medium supports the earlier development and greater expansion of mast cells from fetal liver cells than do media supplemented with serum.

2. Materials and methods

2.1. Antibodies and reagents Biotin-conjugated IgG monoclonal antibody (mAb) against chymase (biotin-B7) and alkaline phosphatase (AP)-conjugated IgG mAb against tryptase (AP-G3) were obtained and prepared as described previously (Irani et al., 1989). IgG mAb anti-Kit, YB5.B8 (Lerner et al., 1991), was a gift from Dr. Leonie K. Ashman (Institute of Medical and Veterinary Science, Adelaide, Australia), and IgG mAb anti-FceRIa, 22E7 (Riske et al., 1991),

were used as described previously (Nilsson et al., 1993). IgG mAb against CD1a NA1 / 34, CD3 UCHT1, CD4 MT310, CD11b 2LPM19c, CD13 ¨ M812, CD14 TUK4, CD15 C3D-1, CD19 HD37 and CD56 T199 were purchased from Dako (Carpinteria, CA, USA). RhSCF was kindly provided by Amgen (Thousand Oaks, CA, USA).

2.2. Culture of human mast cells from dispersed fetal liver Human fetal livers, 16–21 weeks of gestation, were obtained after therapeutic abortions. The experimental protocol was reviewed and approved by the Human Studies Internal Review Board at Virginia Commonwealth University. Each liver was minced finely with scissors, and filtered over a [80 mesh stainless steel sieve. Dispersed cells were layered onto Histopaque 1077 (Sigma, St. Louis, MO, USA) and subjected centrifugation at 5003g for 20 min, collected from the interface, and washed in phosphate-buffered saline (PBS). These mononuclear cells were suspended at 1310 6 cells / ml in Dulbecco’s modified Eagle (DMEM; Sigma), RPMI1640 (Sigma), or AIM-V media (Life Technologies, Rockville, MD, USA) supplemented where indicated with 10% heat-inactivated controlled process serum replacement-3 (CPSR-3; Sigma). In all cases, rhSCF was included at a final concentration of 50 ng / ml. Half of culture medium was changed twice a week, and cells were cultured for up to 12 weeks. Cell viability was determined using 0.05% trypan blue. When the cell number reached 2310 6 cells / ml in a well, half of the cells were split to another well. Mast cell purity was determined by toluidine blue staining, by tryptase immunocytochemical staining of cytocentrifuged preparations, and by flow cytometry with anti-Kit mAb.

2.3. Fluorescence-activated cell sorter ( FACS) analysis Surface expression of Kit and FceRIa, CD1a, CD3, CD4, CD11b, CD13, CD14, CD15, CD19 and CD59 were assessed by flow cytometry. Cells (23 10 5 cells) were incubated in PBS containing 10%

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human AB serum (Sigma) to block FcgR binding of the mAb. Cells were washed in PBS containing 1% fetal calf serum (FCS) and 0.1% sodium azide, incubated with each mAb or control mouse IgG (Sigma) for 1 h at 48C, washed as above, incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulins (Becton-Dickinson, San Jose, CA, USA) for 15 min at 48C, resuspended in sheath solution, and analysed with a FACScan (Becton-Dickinson). HMC-1 cells, a human mast cell leukemia cell line (Butterfield et al., 1988), and KU812 cells, a human basophil leukemia cell line (Kishi, 1985), were used as positive controls for surface Kit and FceRIa expression, respectively. The net percentage of positive cells was calculated by subtracting the percentage of cells labeled with a negative control antibody (set at 3%) from cells labeled with the experimental mAb.

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of 10 mM MES, pH 6.5, containing 2 M NaCl, and sonicated on ice with a model W-225R sonicator and microtip (Heat System-Ultrasonic, Plainview, NY, USA) using 10 pulses twice at power 2.5 with a 50% pulse cycle. Cell-free supernatants were obtained after centrifugation at 12 0003g for 20 min at 48C. Tryptase enzymatic activity was determined spectrometrically by cleavage of 0.1 mM tosyl-Gly-ProLys-p-nitroanilide (Sigma) in 5 mM Hepes, pH 7.6, containing 0.12 M NaCl and 100 mg / ml soybean trypsin inhibitor (Sigma) as described (Xia et al., 1997), where 1 U of enzyme activity cleaves 1 mmol of substrate per min. A specific activity of 20 U / mg was used to estimate the cellular mass of enzymatically active tryptase. Total immunoreactive tryptase levels were measured by ELISA using mAb B12 to capture tryptase and biotinylated mAbs G3 and G4 to detect captured tryptase as described (Schwartz et al., 1994; Ren et al., 1998), purified human lungderived tryptase was used as standard.

2.4. Immunocytochemistry of cell preparation The protease phenotype of the cultured mast cells was assessed in cytocentrifuged preparations by double immunocytochemical labeling with both biotin-B7 and AP-G3 as described (Irani et al., 1989). Staining was visualized with 3-amino-9ethylcarbazole (AEC) in 0.01% H 2 O 2 to stain MC TC cells reddish brown, and with naphthol AS-MX / fast blue RR to stain MC T cells blue. To confirm the results of double immunocytochemical staining, some of the preparations were subjected to single indirect immunocytochemical staining against chymase and tryptase. For chymase detection, horseradish peroxidase-conjugated horse anti-mouse IgG (Vector, Burlingame, CA, USA) was added to B7labeled cells and visualized with AEC in 0.01% H 2 O 2 . For tryptase detection, AP-conjugated rabbit anti-mouse IgG (Sigma) was added to G3-labeled cells and visualized with naphthol AS-BI / new fuchsin to stain cells red. Cells were lightly counterstained with methylene green or hematoxylin.

2.5. Tryptase enzymatic activity and immunoassay measurements Pellets of 1310 6 cells were suspended in 500 ml

2.6. Effect of serum on culture in AIM-V medium To analyze whether serum affects mast cell growth, CPSR-3 was employed as a serum supplement, as described for fetal liver cell cultures in DMEM (Du et al., 1997). In one set of experiments, 10% CPSR-3 was added to media used to culture freshly dispersed fetal liver cells with 50 ng / ml of rhSCF. In a second set of experiments, fetal liver cells cultured in AIM-V medium or in CPSR-3supplemented RPMI-1640 medium, each containing 50 ng / mlof rhSCF for 4 weeks were split and cultured with 50 ng / ml of rhSCF for another week in AIM-V medium with or without 10% CPSR-3 or in RPMI-1640 supplemented with 10% CPSR-3.

2.7. Statistics Mean values were compared by one-way ANOVA. When the ANOVA P value was significant (P, 0.05), post hoc comparisons between control (RPMI1640 with CPSR-3) and experimental groups were determined by a Bonferroni t-test. SigmaStat 2.03 (SPSS, Richmond, CA, USA) was used for these statistical measures.

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3. Results

3.1. Generation of human mast cells from dispersed fetal liver cells in serum-free medium The growth pattern of dispersed fetal liver cells cultured with 50 ng / ml of rhSCF in standard RPMI1640 containing 10% CSPR-3 was compared to that in AIM-V serum-free medium and DMEM containing 10% CPSR-3, as shown in Fig. 1. In AIM-V medium, total cell numbers were dramatically increased compared to serum-containing media from 3 to 6 weeks of culture. The development and survival of mast cells in AIM-V medium, as in serum-supplemented media, is completely dependent on the presence of rhSCF. Cells cultured without rhSCF each of the media died within a few days. By the first week of culture, light microscopy revealed that cells developed in AIM-V medium were more uniform in appearance compared to cells in serum-supplemented media. By day 14, 6466% (mean6S.E.M., n55) of the cells were metachromatically stained with toluidine blue in AIM-V serum-free medium, compared to 3769% of the cells in serum-supplemented DMEM (n55) and 3169% of the cells in RPMI-1640 medium (n55). After 6 weeks, more than 95% of the cells cultured in AIMV media had metachromatic granules (Fig. 1B), compared to 80–90% of the cells in serum-supplemented media. Fibroblast-like cells were attached on the bottom of the culture wells containing serum, but not in the serum-free media. By light microscopy, cytocentrifuge preparations of the cells generated in AIM-V medium had nuclei that were predominantly oval and unsegmented (Fig. 2A), whereas most of the cells in serum-supplemented media had nuclei that were segmented (Fig. 2B). To confirm that metachromatically stained cells developed in AIM-V medium were mast cells, surface Kit expression was examined by flow cytometry. After 4 weeks of culture in AIM-V medium, almost all of the viable cells (98%) expressed surface Kit, compared to 82% in serum-containing media. The increased number of total cells and increased purity of mast cells obtained with serumfree AIM-V media resulted in a dramatic 3–4-fold increase in mast cell number compared to serumcontaining media (Fig. 1C), which was evident by 2

Fig. 1. Total cell numbers (A), percentages of mast cells (B) and numbers of mast cells (C) during the development of fetal liverderived mast cells in different types of media. Fetal liver cells were cultured in AIM-V medium (open circles), DMEM with CPSR-3 (open triangles) and RPMI-1640 medium with CPSR-3 (closed circles) containing 50 ng / ml of rhSCF for the times indicated. When the cell concentration (cells / ml) passed 2310 6 , half of the cells were transferred to another well. This occurred for cells in AIM-V at weeks 1, 2 and 3; for cells in RPMI with CPSR-3 at weeks 1 and 2, and for cells in DMEM with CPSR-3 at week 1. Mast cell numbers were calculated from total numbers of viable cells and the percentages of cells that stained metachromatically with toluidine blue.

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medium tryptase 1 cells accounted for 9662% of total cells (mean6S.E.M., n55). The tryptase staining pattern was granular for mast cells in both serum-free AIM-V (Fig. 2C) and serum-containing (Fig. 2D) media. Tryptase enzymatic activity was measured in cell extracts from 6 week cultures (Fig. 3). The mean tryptase activities from cells cultured in DMEM containing CPSR-3 and in serum-free AIM-V were not significantly different than for cells cultured in RPMI-1640 containing CPSR-3 (P. 0.05). The amounts on a weight basis (mg / 10 6 mast cells) of enzymatically active tryptase calculated per million mast cells (mean6S.D., n53) were 2.260.4 (RPMI), 2.060.4 (DMEM) and 1.860.2 (AIM-V).

Fig. 2. Protease phenotype of fetal liver-derived mast cells developed in different types of media. Toluidine blue staining (A,B) and immunocytochemical staining against tryptase (C,D) of 6-week-old cells, and immunocytochemical staining against chymase (E,F) of 10-week old cells cultured in AIM-V medium (A,C,E) or in 10% CPSR-3-supplemented RPMI-1640 medium (B,D,F) in the presence of rhSCF are illustrated. Closed arrowheads point to examples of mononuclear cells; open arrowheads to examples of cells with deeply divided nuclei. Original magnifications, 3100.

weeks and persisted through 6 weeks of culture. Ultimately, the number of mast cells obtained exceeded the number of fetal liver cells initially placed into culture by 6- to 7-fold. However, in no case did cells express surface FceRI, indicating that the cultured mast cells were functionally immature, as reported previously for fetal liver-derived mast cells generated in serum-supplemented media containing rhSCF (Nilsson et al., 1993; Xia et al., 1997). The protease phenotype of the mast cells was determined by staining cytocentrifuge preparations for tryptase and chymase. In all culture conditions, tryptase 1 cells increased in parallel to those stained metachromatically with toluidine blue and those expressing surface Kit. By 6 weeks in AIM-V

Fig. 3. Cellular levels of tryptase by enzyme activity (A) and immunoassay (B). Mast cells developed in AIM-V medium or in either DMEM or RPMI-1640 medium supplemented with 10% CPSR-3 for 6 weeks were examined. Tryptase was measured in extracts of 1310 6 mast cells (.95% purity). For enzyme activity, tosyl-Gly-Pro-Lys-p-nitroanilide was used as substrate, and data is expressed as mU / 10 6 cells. For immunoassays, total tryptase levels were measured by a sandwich ELISA. In each case data are illustrated as the mean6S.D. values from three independent experiments.

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Total tryptase (mg / 10 6 mast cells) levels by immunoassay were 3.760.1 (RPMI), 3.760.04 (DMEM) and 3.660.04 (AIM-V). The slightly higher amounts of tryptase measured by immunoassay could reflect the presence of enzymatically inactive forms of tryptase in the cell extracts. The percentage of chymase 1 cells developed from fetal liver cells cultured in serum-supplemented RPMI-1640 with rhSCF was reported to be 31% at 6 weeks (Irani et al., 1992a), and only 1% at 4 weeks in serum-supplemented DMEM (Xia et al., 1997). In the present study, cultured mast cells developed in 10% CPSR-3-supplemented RPMI-1640 medium showed faint staining for chymase (Fig. 2F) in 1263% of the cells at 6 weeks (n55). By 12 weeks, chymase 1 cells accounted for 1464% (n55), an insignificant change. Although the percentage of chymase 1 cells in serum-free media at 6 weeks (1362%, n55) was not significantly different from cells in serum-supplemented medium (P.0.05), by 10–12 weeks 53613% (n55) of the cells developed in AIM-V media exhibited an MC TC cell phenotype (Fig. 2E), suggesting AIM-V media without serum permitted greater chymase expression.

3.2. Effect of serum supplement into AIM-V medium on mast cell proliferation To determine whether the increased numbers of mast cells obtained using AIM-V serum-free media were due to AIM-V or to the absence of serum supplement, freshly dispersed fetal liver cells were cultured in AIM-V media with and without 10% CPSR-3, and compared to RPMI-1640 with CPSR-3. Supplementing AIM-V media with serum from day 0 of culture, as with AIM-V media alone, resulted in modest but significantly higher total cell numbers compared to RPMI-1640 with CPSR-3 (Fig. 4A). When two of the four cultures illustrated in Fig. 4A were examined at 6 weeks, total cell numbers in AIM-V media alone (6.2 and 8.2310 6 cells) were similar to those in AIM-V with CPSR-3 (6.4 and 6.9310 6 cells), but substantially higher than in RPMI-1640 with CPSR-3 (1.2 and 1.2310 6 cells). By FACS analysis, numbers of Kit 1 cells were comparable in both sera-containing media at 2 weeks, but were markedly higher (6–7-fold) in serum-free AIM-V media (Fig. 4B,C). However, by 6

weeks of culture, almost all cells were Kit 1 in each culture conditions; the numbers of Kit 1 cells were comparable in AIM-V media alone (5.8 and 7.5310 6 cells) and with CPSR-3 (6.0 and 6.2310 6 cells), being markedly greater than in RPMI-1640 with CPSR-3 (1.0 and 1.1310 6 cells). Of possible interest is the observation that cells developed in serumsupplemented media exhibit stronger surface-staining for Kit than do cells developed in AIM-V alone. Thus, serum supplementation transiently suppresses mast cell development in AIM-V media, but by itself does not account for the lower numbers of cells and mast cells observed with RPMI-1640 containing serum. The expression of various leukocyte surface markers were examined on fetal liver cells cultured for 2 weeks in AIM-V media with and without CPSR-3 and in serum-supplemented RPMI-1640 media. Results are summarized in Table 1. Increased percentages of cells expressing CD11b, CD14 and CD15 were measured in sera-containing media, suggest that serum facilitates the development of non-mast cell lineages. To determine whether serum would also affect survival or proliferation of mast cells later in their development, cells were collected after 4 weeks of culture with rhSCF in serum-free AIM-V media and in 10% CPSR-3-supplemented RPMI-1640 media. Washed cells from each original culture condition were replated at 10 6 cells / ml with rhSCF in AIM-V media with and without CPSR-3, and in RPMI-1640 media with CPSR-3, and cultured for another week. Regardless as to whether the cells were initially cultured in AIM-V media (Fig. 5A) or in CPSR-3supplemented RPMI-1640 media (Fig. 5B), mast cell numbers declined 6 to 12% in CPSR-3-supplemented RPMI-1640, while mast cell numbers increased 16– 46% in AIM-V with or without serum supplementation. These data suggest that AIM-V is more supportive of fetal liver-derived mast cell survival than RPMI-1640, regardless of the presence of CPSR-3.

4. Discussion The current study provides evidence that AIM-V serum-free media improves the yield and purity of

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Fig. 4. Effect of serum supplement on mast cell development in AIM-V media. Freshly dispersed fetal liver cells were cultured with 50 ng / ml of rhSCF in AIM-V with and without 10% CPSR-3 and in RPMI-1640 medium with 10% CPSR-3. (A) Total numbers of viable cells. Bars show mean6S.D. values from four independent experiments after 2 weeks. Trypan blue was used to assess viability. Mean numbers of cells obtained in AIM-V alone and with CPSR-3 were significantly higher than in RPMI-1640 with CPSR-3. (B) Kit expression. Cells were assessed by flow cytometry after 2 and 6 weeks of culture, in each case at 4 days after feeding. Dead cells were excluded from analysis by propidium iodide staining. The figure is representative of four separate experiments at 2 weeks, and two separate experiments at 6 weeks. (C) Mast cells. The numbers of Kit 1 mast cells were calculated from the percentage of Kit 1 cells measured by flow cytometry after 2 weeks of culture. Dead cells were excluded by propidium iodide staining. Bars show mean6S.D. values from four independent experiments. The mean number of mast cells obtained in AIM-V alone was significantly (P,0.001) higher than in RPMI-1640 with CPSR-3.

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Table 1 Surface marker expression on fetal liver cells (net % positive) developed for 2 weeks in serum-free and serum-supplemented media a Cell-surface marker

CD11b CD13 CD14 CD15 Kit

Cell culture medium AIM-V

AIM-V/ CPSR-3

RPMI / CPSR-3

6.6 57 ,1 13 53

24 60 4.8 45 7.8

11 21 4.7 18 9.7

a The net percent of cells expressing surface CD1a, CD3, CD19, CD59 or FceRIa, regardless of the media used, was less than 1%.

human mast cells derived from fetal liver cells in the presence of rhSCF compared to more commonly used RPMI-1640 and DMEM. The first report in 1994 of a human mast cell culture in serum-deprived medium (Iscove’s modified Dulbecco’s medium) required the use of both SCF and IL-3, and resulted in mast cells derived from CD34 1 cord blood cells after approximately 2 months of culture (Durand et al., 1994). However, these cells were mostly Kit 2 by flow cytometry, and died in the absence of IL-3, uncharacteristic properties of human mast cells. More recently, human mast cells were developed from cord blood cells in serum-free a-medium supplemented with rhSCF, but required hypoxic conditions (5% O 2 ) (Kinoshita et al., 1999). The present study utilized AIM-V medium at an atmospheric concentration of oxygen (21%) and 6% CO 2 , which proved to be effective for generating human mast cells from fetal liver cells. Preliminary data indicate similar results using cord blood-derived progenitors. With SCF as the only exogenously added growth factor, mast cells developed more rapidly in the absence than in the presence of a serum supplement. Serum may contain factors that delay the development of mast cells, and that encourage the development of other cell types, as evidenced by higher numbers of cells expressing surface CD11b, CD14 and CD15, myeloid markers not typically found on mast cells, after 2 weeks of culture in serum-supplemented media. Once mast cells had developed in serum-free AIM-V medium, addition of serum did

Fig. 5. Effect of media type on mast cells derived from fetal liver progenitors. Four-week-old mast cells developed in AIM-V medium (9162% purity) (A) or in CPSR-3-supplemented RPMI1640 medium (68610% purity) (B) were collected, washed in PBS, and replated at 1310 6 cells / ml into AIM-V medium with and without 10% CPSR-3 or into RPMI-1640 medium with 10% CPSR-3. Cells were cultured for another week in the presence of 50 ng / ml rhSCF, and viable mast cell numbers were measured using trypan blue and toluidine blue. The mean percent mast cell purities after 1 week of culture in AIM-V, AIM-V/ CPSR-3 and RPMI-1640 / CPSR-3, respectively, were 98, 96 and 95 when the original culture medium was AIM-V, and 79, 88 and 93 when the original culture medium was RPMI-1640 / CPSR-3. Data shows mean6S.D. values from four independent experiments. Regardless of the media used during the initial 4 weeks of culture, both AIM-V with or without CPSR-3 resulted in significantly greater numbers of mast cells than did RPMI-1640 with CPSR-3.

not appear to affect mast cell survival or proliferation. AIM-V medium appeared to facilitate the development of chymase-positive cells from our suspension cultures of fetal liver cells. Previously, MC T cells were the predominant type seen in suspension cultures utilizing either RPMI-1640 supplemented with serum (Irani et al., 1992a) or DMEM supplemented with serum (Xia et al., 1997), as well as in cocultures with mouse 3T3 fibroblasts using RPMI-

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1640 supplemented with serum (Irani et al., 1992b). Given that SCF-dependent fetal liver-derived mast cells after 4 weeks of suspension culture contain abundant amounts of chymase mRNA but little if any chymase protein (Xia et al., 1997), the current serum-free culture condition may be more permissive for expression of chymase protein in those cells that also express chymase mRNA. Also of note is the finding that the rounded nuclear morphology observed for mast cells obtained in serum-free AIM-V is more typical of tissue-derived mast cells, and differs from the multi-lobed nuclei often observed in serum-containing media. In summary, serum-free AIM-V medium, compared to serum-supplemented RPMI-1640 or DMEM, supplemented with rhSCF substantially increased the number of mast cells obtained from fetal liver cells, resulted in the earlier development of mast cells, a nuclear morphology more typical of tissue-derived mast cells and better expression of chymase protein.

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