Culture of Intestinal Mast Cells

In the absence of growth factors, MACS-enriched mast cells die completely within 3-7 d. Mast cell survival is slightly prolonged if nonenriched cell fractions are cultured, which is related to growth factors provided by contaminating cells. Long-term culture of human intestinal mast cells can be achieved in the presence of recombinant SCF preventing mast cell apoptosis and inducing proliferation (15,19). Additional factors such as IL-3 and IL-4 enhance mast cell growth in the presence of SCF by decreasing apoptosis (IL-3) or increasing proliferation (IL-4, Fig. 2 and Table 1). In the absence of SCF, IL-4 has no effect and IL-3 has only a minimal effect on mast cell survival. The addition of IL-3, IL-4 or IL-3 + IL-4 to the culture medium in combination with SCF can enhance mast cell numbers after 2-3 wk of culture approximately two-, three-, or fourfold, respectively, in comparison with mast cells cultured with SCF alone (16,20).

The purity of mast cells largely increases during culture. If mast cells were enriched by MACS before culture, purity is generally 85-95% after 1 wk and 95-100% after 2 wk of culture (15). If nonenriched cell fractions were cultured

Fig. 2. Mast cell recovery after culture of MACS-enriched human intestinal mast cells. Mast cells were cultured for indicated times in the presence of SCF (50 ng/mL), SCF + IL-3 (100 ng/mL), SCF + IL-4 (2 ng/mL), or SCF + IL-4 + IL-3.

Table 1

Effects of IL-3, IL-4, and TGF-P on Human Intestinal Mast Cells

Cytokine IL-3 IL-4 TGF-P

Mast cell numbers

Table 1

Effects of IL-3, IL-4, and TGF-P on Human Intestinal Mast Cells

Mast cell numbers

after 21 d of culturea

150-250%

200-400%

20-50%

Proliferationa

T

i

Apoptosisa

i

^

T

Histamineab

T

T

i

LTC4/D4/E4ab

T

T

i

PGD2ab

^

T

TNF-aab

n.t. d

i

IL-6ab

n.t. d

i

n.t. d

IL-3, IL-5, IL-13 abc

n.t. d

T

i

Ratio of MCt/MCtc a

n.t. d

T

i

a Phenotype of mast cells cultured in the presence of SCF alone is compared with the phenotype of mast cells cultured in the presence of SCF + the indicated cytokine.

b Mediator release upon FceRI-crosslinking.

c IL-4 induces small amounts of IL-5 even without further stimulation. d Not tested.

PGD2, prostaglandin D2; LT, leukotrienes; TNF-a, tumor necrosis factor alpha; IL, interleukin; MC, mast cell; T, tryptase; TC, tryptase + chymase; TGF-ß, transforming growth factor ß.

a Phenotype of mast cells cultured in the presence of SCF alone is compared with the phenotype of mast cells cultured in the presence of SCF + the indicated cytokine.

b Mediator release upon FceRI-crosslinking.

c IL-4 induces small amounts of IL-5 even without further stimulation. d Not tested.

PGD2, prostaglandin D2; LT, leukotrienes; TNF-a, tumor necrosis factor alpha; IL, interleukin; MC, mast cell; T, tryptase; TC, tryptase + chymase; TGF-ß, transforming growth factor ß.

Fig. 3. Influence of culture on histamine and sLT (LTC4/D4/E4) release after FceRI-crosslinking. Mast cells were stimulated with for 30 min with the MAb 22E7 (100 ng/ mL) directly after MACS purification or after 3, 7, or 14 d of culture in the presence of SCF (50 ng/mL). Specific histamine release as a percentage of total histamine (determined after cell lysis) is shown.

Fig. 3. Influence of culture on histamine and sLT (LTC4/D4/E4) release after FceRI-crosslinking. Mast cells were stimulated with for 30 min with the MAb 22E7 (100 ng/ mL) directly after MACS purification or after 3, 7, or 14 d of culture in the presence of SCF (50 ng/mL). Specific histamine release as a percentage of total histamine (determined after cell lysis) is shown.

in the presence with SCF, mast cell survive selectively and highly purified mast cells can be obtained after 4-5 wk of culture. Although 95-100% pure mast cells can be obtained in some cultures, the purity often is poorer because of contaminating fibroblasts. In our hands, IL-4 does not induce the growth of intestinal lymphocytes and improve the purity of mast cells cultures used in combination with SCF (see Notes 11 and 12).

Cultured mast cells release much higher amounts of mediators in response to Fce receptor I (FceRI) crosslinking than freshly isolated mast cells (see Subheading 3.4. and Fig. 3), which is most likely related to the fact that the isolation and purification procedure causes a reversible damage of the cells that reduces their functional capacities. During culture, mast cells regain the full capacity to respond to FceRI crosslinking. This suggests that cultured mast cells reflect more accurately the phenotype of mast cells in vivo than freshly isolated mast cells (15,16,19).

Human intestinal mast cells also can be maintained in co-culture with human endothelial cells or human fibroblasts. Co-culture with endothelial cells requires direct cell contact. The survival and proliferation of intestinal mast cells are mediated by at least two pathways: (1) the interaction between transmembrane SCF expressed by endothelial cells and c-kit on mast cells; and (2) the interaction between the adhesion molecules and very late activation antigen-4, expressed by mast cells and vascular cell adhesion molecule-1 induced on endothelial cells in the presence of mast cells (21). Human intestinal fibroblasts prevent mast cell apoptosis but do not induce proliferation. In contrast to findings with the rodent 3T3-fibroblast cell line, human intestinal fibroblasts mediate mast cell survival independent of SCF by a yet unknown soluble factor (22). Both co-culture systems are useful in studying the interaction of mast cells with the respective cell type; however, they are insufficient techniques if one wishes to obtain high numbers of purified intestinal mast cells.

1. Culture mast cells in culture medium. Adjust the cell concentration to 1-2 X 105 mast cells/mL for MACS-enriched cells or to 2 X 106 of total cells/mL for unpurified cells. Culture of intestinal mast cells can be performed in 96-, 48-, 24-, 12-, or 6-well flat-bottomed plates in 0.2, 0.5, 1, 2, or 5 mL of cell culture medium, respectively.

2. Add SCF at a concentration of 50 ng/mL. If required add other cytokines (see Table 1 and Note 12).

3. Maintain cells in a humidified atmosphere containing 5% CO2 at 37°C.

4. Change 50% of the culture medium twice during the first week and then once a week thereafter. Add new growth factors each time.

5. Subculture cells if required (see Note 13).

6. After an appropriate culture period (see text above), harvest mast cells using a cell scraper. Count the cells after staining with trypan blue. Prepare cytocentrifuge smears and stain with May-Grunwald/Giemsa to perform a differential count.

We have been using standard assay for the analysis of proliferation (Ki-67, BrdU-, and 3H-thymidin incorporation) and apoptosis (annexin V, caspase assays [16,20,22]). These assays are used frequently and they will not be described here. For all assays, use only mast cells that have been cultured for at least 4-7 d. Many mast cells die within the first days of culture because of damage caused by the isolation and MACS procedure (Fig. 2). Proliferation starts after approx 4 d (16). Short-term effects can be assayed by using precultured mast cells.

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