Isolation and Culture of HISM Cells 417

32
Isolation, Culture, and Characterization of Human
Intestinal Smooth Muscle Cells
Martin Graham and Amy Willey

1. Introduction
In vitro models utilizing human mesenchymal cells isolated from normal
and pathological tissue have proven very useful for studies of wound repair
and fibrosis. We have been studying the pathogenesis of intestinal fibrosis and,
over the course of 20 yr, have refined techniques for the isolation, culture, and
characterization of smooth muscle cells of the human intestinal muscularis propria
(1). These methodologies have been adapted for isolation of similar cell types from
liver, gallbladder, and blood vessels (2). In this chapter, we describe techniques
for the isolation and culture of these cells (procedure A), for the quantitation
of procollagen secretion (procedure B), and for the screening of isolates for the
expression of smooth muscle–specific cytoskeletal proteins (procedure C).

2. Materials
2.1. Procedure A: Isolation and Culture of Human Intestinal
Smooth Muscle Cells
1. Tissue culture plates.
2. Dulbecco’s modified Eagle’s medium (DMEM).
3. Nystatin solution: 10,000 U/mL of stock.
4. Penicillin/streptomycin solution (Pen/Strep): 10,000 U/mL of stock.
5. Supplemented DMEM-0: Each 50 mL of DMEM contains 2.5 mL Pen/Strep
stock and 0.25 mL of Nystatin stock.
6. Sterile scissors and forceps.
7. Sterile filter paper: Whatman #1 cut in a circle to fit on the stage of a tissue
slicer.
8. Tissue slicer (cat. no. 6727C10; Thomas Scientific, Swedesborough, NJ).

From: Methods in Molecular Medicine, vol. 78: Wound Healing: Methods and Protocols
Edited by: Luisa A. DiPietro and Aime L. Burns © Humana Press Inc., Totowa, NJ

417
418 Graham and Willey

9. Collagenase solution: 0.5 mg/mL culture medium, sterile filtered (see item 10
and Note 1) (cat. no. 4197; Worthington, Freehold, NJ).
10. Syringe filter (0.2 μm) (Acrodisc; Fisher, Pittsburgh, PA).
11. Conical tubes (50 mL).
12. Fetal bovine serum (FBS) (see Note 2).
13. DMEM supplemented with 10% FBS (DMEM-10).

2.2. Procedure B: Analysis of Procollagen Secretion by Slab Gel

Electrophoresis of Radiolabeled Proteins in Culture Medium
1. Tissue culture plates.
2. DMEM-0.
3. FBS (see Note 2).
4. DMEM-10.
5. L-Ascorbate (Sigma, St. Louis, MO): Make fresh in DMEM at 100 μM.
6. [3,4-3H]-Proline (Amersham, Piscataway, NJ).
7. Glacial acetic acid.
8. Isopropanol.
9. 2X Laemmli’s buffer (3).
10. Bio-Rad minigel apparatus.
11. Molecular weight marker (cat. no. RPN756; Amersham).
12. 1 M Sodium salicylate enhancing solution.
13. X-ray film (Hyperfilm MP, cat. no. RPN1677L; Amersham).

2.3. Procedure C: Screening of Human Intestinal Smooth Muscle

Cells for Smooth Muscle–Specific Cytoskeletal Proteins
1. Tissue culture plates.
2. DMEM-0.
3. FBS (see Note 2).
4. DMEM-10.
5. RIPA buffer: 1X phosphate-buffered saline, 1% Igepal CA-630 (Sigma), 0.5%
sodium deoxycholate, and 0.1% sodium dodecyl sulfate.
6. 21-Gauge needle.
7. Antibodies:
a. α–Smooth muscle isoactin: 42 kDa (Sigma A2547).
b. h-Caldesmon: 150 kDa (Sigma C4562).
c. Vinculin/metavinculin: 116 kDa (Sigma V9131).
d. Tropomyosin: 36–39 kDa (Sigma T2780).

3. Methods
3.1. Procedure A: Isolation and Culture of Human Intestinal
Smooth Muscle Cells
Intestinal tissue harvested in the operating room should be kept on ice in a
sterile container and processed on the same day.
Isolation and Culture of HISM Cells 419

1. Filet open the specimen and place the tissue, mucosa side up, in a 100-mm
tissue culture plate containing 20 mL if supplemented DMEM. If the specimen is
contaminated with fecal material, wash three times in this medium solution.
2. Remove the mucosa from the muscularis using sharp, pointed scissors in the
cleavage plane of the submucosa.
3. Cut the muscularis into 2 × 2 cm squares and place, adventitia side down, on
wet, sterile filter paper covering the tissue slicer stage.
4. Slice through the specimen with the blade in the plane of the muscularis, moving
from submucosa through to serosa.
5. Discard the initial submucosal and last serosal slices.
6. If desired, culture the innermost circular layer separately from the outer longitu-
dinal layer. The two layers can be differentiated by the difference in grain as
viewed from above.
7. Place the sliced tissue in 20 mL of collagenase solution in a clean 100-mm
tissue culture plate. Mince with sterile scissors and incubate overnight at 37°C
in 8% CO2.
8. Place the contents of the culture dish in a 50-mL conical tube, add an equal
volume of DMEM-10, and centrifuge at 100g for 3 min.
9. Remove the supernatant, and resuspend the pellet in 15 mL of DMEM-10. Place
5-mL aliquots of the suspension in three to four 100-mm culture plates and
incubate at 37°C in 8% CO2.
10. Allow the cultures to sit for a minimum of 48 h without disturbance.
11. Carefully aspirate the culture medium, allowing the tissue and cells to remain
undisturbed in the plate. Add 5 mL of DMEM-10 to the plate. Again, take care not
to disturb the tissue and cells. Repeat medium replacement every 3 d. Individual
cells that have been released from the tissue will adhere to the plate, begin to
spread, and then begin to proliferate. Small pieces of tissue that have adhered to
the dish will begin to sprout cells.
12. The culture plate should be confluent with cells after 4 wk of culture. When
cells begin to “ridge” on the bottom of the plate, passage and expand the cells
(see Note 1).

3.2. Procedure B: Analysis of Procollagen Secretion by Slab Gel

Electrophoresis of Radiolabeled Proteins in Culture Medium
1. Grow cells to confluence in 100-mm culture dishes in culture medium containing
10% FBS (see Notes 3–5).
2. Rinse plates two times with 5 mL of DMEM-0 and then add 5 mL of DMEM-0
with fresh L-ascorbate. Incubate overnight.
3. Remove the medium and replace with 5 mL of DMEM-0 containing fresh
L-ascorbate and 100 μCi of [3,4-3H]-proline. Incubate for 24 h.
4. Transfer the medium to a tube on ice. Rinse the plate with 5 mL of DMEM-0. Add
rinse to the tube. Add glacial acetic acid to achieve a concentration of 0.5 N.
5. Scrape the cells off of the plate in 5 mL of 0.5 N acetic acid into a separate
tube at 4°C.
420 Graham and Willey

Fig. 1. Example of autoradiogram of procollagen secreted by HISM cells in vitro.

HISM cells were incubated with [3,4-3H]-proline for either 6 or 24 h in the absence
(control) or presence (asc) of ascorbate. Media were harvested and processed as
described in Subheading 3.2. The specific upregulation of procollagen secretion
induced by ascorbate is seen. Molecular weight markers are shown on the left.
(Reprinted with permission from ref. 7.)

6. Mix the medium and cell tubes well and incubate at 4°C overnight. Process
separately as below in 7–14.
7. Add an equal volume of isopropanol to each tube, vortex, and place at –20°C
for 20 min.
8. Centrifuge at 7500g at 4°C for 30 min.
9. Remove the supernatant and dry the pellet in an evaporator or under a stream
of nitrogen.
10. Add 100 μL of 2X Laemmli’s buffer.
11. Vortex and heat to 100°C for 1 min until the protein is solubilized.
12. Run 20 μL of each sample on 1.5-mm-thick 5% acrylamide gel in the minigel
apparatus until the 66 kDa molecular weight marker is at the bottom of the gel.
Each well should have approx 20,000 dpm for an adequate signal of procollagen
bands. The radioactivity of the samples should be determined prior to running
the gel in order to facilitate sample loading.
13. Fix the gel and enhance in sodium salicylate solution.
14. Dry the gel and expose to X-ray film overnight at –80°C. Then quantitate
procollagen bands by densitometry (Fig. 1) (4–7).

3.3. Procedure C: Screening of Human Intestinal Smooth Muscle

Cells for Smooth Muscle–Specific Cytoskeletal Proteins
To ensure that the clone of cells isolated from the muscularis is phenotypi-
cally “true,” it is important that screening for the expression of smooth muscle-
Isolation and Culture of HISM Cells 421

specific proteins be performed with each isolate. Using the aforementioned

isolation and culture techniques, we find that less than 1 in 20 isolates will not
express smooth muscle–specific proteins. Those isolates are then discarded.
The technique involves Western blotting of human intestinal smooth muscle
(HISM) cell lysates for α–smooth muscle isoactin, h-caldesmon, metavinculin,
and tropomyosin. Since publication of work with α–smooth muscle isoactin
(8), we have extended those observations to demonstrate that both h-caldesmon
and metavinculin are expressed in vitro by HISM cells. Cells not expressing
these proteins are, therefore, considered to have “de-differentiated” in culture.

1. Plate 104 HISM cells in a 60-mm culture plate in DMEM-10 (see Note 3).
2. Allow to grow to confluence. Do not change the medium for 5 d prior to
harvest.
3. Rinse the plates two times with 5 mL of DMEM-0. Incubate for 5 h and remove
the medium.
4. Scrape the cells into RIPA buffer and force through a 21-gage needle.
5. Quantitate the protein in the lysate.
6. Perform immunoblotting on a Bio-Rad minigel apparatus loading 20 μg of
protein/well and utilizing the primary antibodies detailed in Subheading 2.3.,
item 7.

Figure 2 demonstrates a typical screen performed on six lines of HISM

cells with a line of human dermal fibroblasts as control. Figure 2 demonstrates
that the fibroblast line does not express any of the markers but does express
vinculin. One HISM cell line (lane 4) did not express either metavinculin or
h-caldesmon and was considered to have de-differentiated.

4. Notes
1. For the isolation technique, different batches of collagenase should be screened
for optimal yield and cell viability. Once a batch is identified as providing a high
yield of viable cells, it should be purchased in bulk and stored.
2. For cell culture, different batches of high-quality, low-endotoxin serum should
be screened for optimal growth rates. Again, once a batch has been identified as
providing good cell growth, it should be purchased in quantity and stored. The
importance of high-quality serum cannot be overemphasized.
3. The plating density of cells is critical for efficient growth to confluence; 10–20
× 103 cells/cm2 is usually sufficient. Cells plated at too low a density will not
proliferate.
4. HISM cells have the fortuitous property, when confluent, of remaining in robust
condition for up to 10 d in culture medium without serum (9). Therefore, for
individual experiments of agonist activity, cells should be grown to confluence
in medium containing 10% FBS and then placed in serum-free DMEM for
24 h prior to running the experiment. Under these conditions, HISM cells are
422 Graham and Willey

Fig. 2. Screening for expression of smooth muscle–specific cytoskeletal proteins

by immunoblotting of cell lysates. Each panel is a separate immunoblot performed
with the antibody denoted on the right. Molecular weight markers are shown on the
left. Lanes 1–5, five different HISM cell lines; lane 6, control line of human dermal
fibroblasts. Note that the fibroblast line does not express any of the markers but
does express vinculin. In lane 4 the HISM cell line did not express metavinculin or
h-caldesmon and was considered to have de-differentiated.

quiescent, but responsive (9). This technique is important because it allows the
investigation of agonists without the contaminating influence of serum, which
has relatively high levels of mitogens (platelet-derived growth factor), cytokines
(transforming growth factor-β), and other inflammatory mediators.
5. Each isolate should be screened for the expression of smooth muscle cell
cytoskeletal markers prior to use (see procedure C, Subheading 3.3.).

Acknowledgment
This work was supported by National Institutes of Health grant DK34151.

References
1. Graham, M. F., Diegelmann, R. F., Elson, C. O., Bitar, K. N., and Ehrlich, H. P.
(1984) Isolation and culture of human intestinal smooth muscle cells. Proc. Soc.
Exp. Biol. Med. 176, 503–507.
2. Sanyal, A. J., Contos, M. J., Yager, D., Zhu, Y., Willey, A., and Graham, M. F.
(1998) Development of pseudointima and stenosis after transjugular intrahepatic
Isolation and Culture of HISM Cells 423

portosystemic shunts: characterization of cell phenotype and function. Hepatology

28, 22–31.
3. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227, 680–685.
4. Graham, M. F., Willey, A., Adams, J., Yager, D., and Diegelmann, R. F. (1996)
Interleukin 1 beta down-regulates collagen and augments collagenase expression
in human intestinal smooth muscle cells. Gastroenterology 110, 344–350.
5. Graham, M. F., Willey, A., Adams, J., and Diegelmann, R. F. (1995) Corticosteroids
increase procollagen gene expression, synthesis, and secretion by human intestinal
smooth muscle cells. Gastroenterology 1091, 454–461.
6. Graham, M. F., Willey, A., Adams, J., Yager, D., and Diegelmann R. F. (1995)
Role of ascorbic acid in procollagen expression and secretion by human intestinal
smooth muscle cells. J. Cell Physiol. 162, 225–233.
7. Rosenblat, G., Willey, A., Zhu, Y. N., Jonas, A., Diegelmann, R. F., Neeman, I., and
Graham, M. F. (1999) Palmitoyl ascorbate: selective augmentation of procollagen
mRNA expression compared with L-ascorbate in human intestinal smooth muscle
cells. J. Cell Biochem. 73, 312–320.
8. Graham, M. F., Gluck, U., Shah, M., and Ben-Ze’ev, A. (1994) β-tropomyosin
and α-actin are phenotypic markers for human intestinal smooth muscle cells in
vitro. Mol. Cell. Diff. 2(1), 45–60.
9. Graham, M. F., Bryson, G. R., and Diegelmann, R. F. (1990) Transforming growth
factor-β1 selectively augments collagen synthesis by human intestinal smooth
muscle cells. Gastroenterology 99, 447–453.