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J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Dia ,side effects
DOI 10.1007/978-0-387-87429-6_33, © Springer Science+Business Media, LLC 2011
Abstract Experimental quality is directly proportional to cell quality. This might not be a valid mathematical equa-tion, but it very succinctly and elegantly describes the work of biologists from all fields of rearch: molecular biology or cell biology, proliferation or apoptosis assays, patch clamp electrophysiology or flow cytometry. One element lies at the heart of the success of each of the procedures: viable and healthy cells. Many days and weeks of valuable experimentation time have been spent perfecting cell isola-tion techniques, simply to guarantee that we can gather reli-able data. Although it is possible to purcha cells from various sources, most rearch groups have developed their own techniques for isolating cells from tissues, techniques which can be adapted relatively easily from one tissue type to another. In addition, although it is always desirable to u freshly isolated cells (less than 8 h after isolation), many investigators have turned to cell culture as a viable a lternative to freshly dissociated cells, although this may prent some scientific challenges and dilemmas. The current c hapter address the isolation of pulmonary artery smooth m uscle cells (PASMCs) and pul
monary artery endothelial (PAECs), particularly from humans, rats, and mice. Generally speak-ing, the methods we outline can also be applied to PASMCs and PAECs from other species, although some modifica-tions may be required. Readers are advid to consult the extensive literature to identify the technique most applicable to their needs.
Keywords Cell preparation • Tissue disction • Pulmonary artery • Freshly dissociated cells • Primary culture • Cell physiology
1 I ntroduction
Experimental quality is directly proportional to cell quality. This might not be a valid mathematical equation, but it very succinctly and elegantly describes the work of biologists from all fields of rearch: molecular biology or cell biology, pro-liferation or apoptosis assays, patch clamp electrophysiology or flow cytometry. One element lies at the heart of the success (and reproducibility!) of each of the procedures: viable and healthy cells. Many days and weeks of valuable experimenta-tion time have been spent perfecting cell isolation techniques, simply to guarantee that we can gather reliable data. Although it is possible to purcha cells from various sources (e.g., Lonza, Invitrogen, and ATCC in North America), most rearch groups have develope
d their own techniques for iso-lating cells from tissues, techniques which can be adapted relatively easily from one tissue type to another. In addition, although it is always desirable to u freshly isolated cells (less than 8 h after isolation), many investigators have turned to cell culture as a viable alternative to freshly dissociated cells, although this may prent some scientific challenges and dilemmas. The current chapter address the isolation of pulmonary artery smooth muscle cells (PASMCs) and pulmo-nary artery endothelial (PAECs), particularly from humans, rats, and mice. Generally speaking, the methods we outline can also be applied to PASMCs and PAECs from other species, although some modifications may be required. Readers are advid to consult the extensive literature to
identify the technique most applicable to their needs.2 C ell Isolation Theory and U
of Enzymes for Tissue Dissociation and Cell Harvesting
Enzymes are the primary tools in tissue culture rearch inso-far as it concerns the isolation of cells and dissociation of intact tissue. Despite the widespread u of enzymes such as collagena, elasta, papain, etc., their mechanisms of action
C.V . Remillard (*)
Division of Pulmonary Critical Care Medicine,母亲节快乐英文
University of California, San Diego, 9500 Gilman Drive, MC 0725, San Diego, CA 92093, USA e-mail:
Chapter 33
Isolation and Culture of Pulmonary Vascular Smooth Muscle and Endothelial Cells
Carmelle V. Remillard, Ayako Makino, and Jason X.-J. Yuan
486 C.V. Remillard et al. in various preparations are poorly understood. As a result, the
choice of one technique or enzyme over another is arbitrary
and bad on experience rather than scientific theory. As
mentioned already, the ultimate goal of cell isolation is to
yield viable, functional dissociated cells. A number of factors
which can determine the outcome of the cell isolation proce-
dure. The are outlined in Table 1. Of the, only the type of tissue and species and the age of the animal are not a mat-ter of choice. The other six parameters are highly variable and can greatly influence cell quality; as such, the are often manipulated to optimize individual cell isolation protocols.
If one examines the literature for isolation techniques for a particular cell type (e.g., PASMCs), the results are varied becau of individual experience with different enzyme combi-nations. Even manuals offer conflicting information, mostly due to a combination of (1) lack of information about the struc-ture of the extracellular matrix and (2) the prence of unknown impurities in the crude enzyme preparations. Although we, as investigators, cannot control the ultimate purity of individual enzyme preparations (we rely on reputable companies to over-e that task), we can inform ourlves about the extracellular matrix components for our tissues of interest.
3 T issue Collection and Isolation
of Vascular Myocytes
3.1 C ollection of Lung Tissue Samples
Human lung tissue samples can be obtained from patients undergoing lung transplantation, lobectomy, and biopsy. Typically, a small (approximately 1 cm3) piece of the periph-eral lung tissues is removed from the lung and placed in cold (4°C) saline until disction can be performed (within 3 h of transplantation). For mice and rats, lungs and heart are removed en bloc following euthanasia (decapitation or cervical disloca-tion are ideal to reduce trauma to the lungs), and placed in warm N-(2-hydroxyethyl)piperazine-N¢-ethanesulfonic acid buffered saline solution (HBSS). Once the tissues have been obtained, fat, blood, and connective tissue are removed using aptic techniques so that vascular tissues can be isolated.3.2 P reparation of Primary Culture PASMCs There are two general methods for the preparation of pri-mary cultures of vascular smooth muscle cells. One involves the direct isolation of cells from enzymatically digested ves-ls and the other involves isolating cells that migrate out from small tissue samples. Since enzyme dissociation is the most commonly ud approach, we will describe procedures that have successfully yielded viable healthy human, mou, and rat PASMCs.
The vascular wall is organized into three layers: an inner layer compod of endothelial cells, a middle layer compod of smooth muscle cells, and an outer adventitial layer com-pod of fibroblasts, connective tissues, and nerve endings. (Fig. 1) The smooth muscle cells that make up th
e medial layer are involved in process such as vascular growth and repair, respon to injury, and vascular contraction.
For the enzymes to reach the medial PASMCs and to iso-late pure smooth muscle cells, the first steps of PASMC isola-tion involve removing the adventitia and intima (Fig. 2a, b). The vesl is placed in fresh HBSS, carefully stretched and pinned onto a discting dish. The adventitia (which mainly contains fibroblasts) can be discted away from the media with forceps. We have also found that a brief treatment (approximately 10 min) with collagena can significantly “loon” the adventitia so that it can be “peeled” off the vesl. After removal of the adventitia, the vesl is cut open longitu-dinally so the endothelial layer can be gently scraped away.
The pulmonary artery, now mostly vascular media, is then cut into pieces and placed in collagena–HBSS (Fig. 2c) at 37°C for 20 min, after which the tissue is allowed to recover overnight for 10–12 h at 37°C in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with fetal bovine rum (FBS), penicillin/streptomycin, and l-glutamine. The vesl is then transferred to a fresh enzyme solution contain-ing collagena, elasta, and albumin, and is incubated at
Table 1Parameters affecting cell isolation outcome
1.Type of tissue
2.Species of origin
3.Age of the animal
4.Dissociation medium ud
5.Enzyme(s) ud
6.Impurities in crude enzyme preparation ud
7.Concentrations(s) of enzymes ud
8.Temperature
9.
Incubation time
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Fig. 1Structure of the pulmonary arteries. Cartoon description of the
pulmonary vasculature starting from the whole lung and at successive
magnifications. Clo-up structure identifies the three main layers of
the artery: the intima, containing pulmonary artery endothelial cells
(PAEC), the media, containing pulmonary artery smooth muscle cells
(PASMC), and the adventitia, compod primarily of fibroblasts (PAFB)
and fibers such as collagen and elastin
487
33 Isolation and Culture of Pulmonary Vascular Smooth Muscle and Endothelial Cells 37°C for approximately 50 min. Finally, the tissue is gently triturated to relea cells (Fig. 2d ) and DMEM (containing 20% FBS) is added to halt digestion. Cells are parated from tissue debris by filtration through a nylon mesh. The enzyme-containing cell suspension is then centrifuged, and the pellet containing the PASMCs is resuspended in fresh 10% FBS–DMEM. Dissociated cells can now be refrigerated for immediate u, or plated in tissue culture dishes contain-ing prewarmed growth medium. For rat PASMCs, we u 10% FBS–DMEM. For human PASMCs, we u a smooth muscle growth medium (SMGM; Lonza) which contains
10% FBS, insulin, human epidermal growth factor , human fibroblast growth factor B, gentamicin, and amphotericin–B. Cells are incubated at 37°C for further growth. The cell medium should be changed initially after 24 h and every 48 h subquently.
accidentallyAlthough we have placed emphasis on DMEM and SMGM, veral media are available to support SMC growth, including Eagle’s minimal esntial medium), medium 199 with Earle’s or Hank’s salt (M199), and Ham’s F12. Many rearchers now u plastic, rather than glass, dishes to plate smooth muscle cells. Cell attachment to plastic dishes or coverslips may be enhanced by coating the surface with fibronectin, vitronectin, laminin, collagen, or lysine [1–3].
3.3 P reparation of Primary Culture PAECs
As with PASMCs, there are a number of isolation techniques which can yield viable endothelial cells. Probably the sim-plest technique is to recover endothelial cells by rubbing them off the intimal surface of the pulmonary arteries. In this technique (Fig. 3a ), the isolated and cleaned pulmonary artery is excid, then inverted to expo the intimal layer. Endothelial cells are obtained by gently scraping the intimal layer with a sterile scalpel or a plastic cell lifter or scraper. Harvested cells are then plated with culture medium (e.g., F12 nutrient mixture, DMEM) supplemented with 10–20% FBS and antibiotics [4].
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The latter technique can be ud only with main pulmo-nary artery gments; however, King et al. [5] have since described a modified method to isolate rat microvascular PAECs (PMVECs). In this technique (Fig. 3b ), thin strips are removed from the lung periphery adjacent to the pleural sur-face, finely minced, and treated with a collagena (type 2, Worthington) and bovine rum albumin–DMEM at 37°C. After approximately 15 min, the mixture is filtered through a sterile 80-mesh sieve and centrifuged to pellet PMVECs. PMVECs are resuspended with complete medium compod of one part microvascular conditioned medium and three parts incomplete medium [80% RPMI, 20% FBS, heparin, Endogro (Vec Technologies), and penicillin–streptomycin]. The centrifugation–resuspe
nsion process is repeated once and the final cell pellet is resuspended in complete medium and allowed to rest at 37°C prior to plating. Finally, PMVEC suspension drops are placed on coverslips, and the cells are allowed to ttle and attach for 1 h at 37°C (5% CO 2, 95% O 2) before complete medium is added.
As shown above, enzymatic dissociation is effective for PMVEC isolation. However, endothelial cells from the main pulmonary artery can also be isolated using enzymes, although the process itlf is somewhat different from that for PASMCs or PMVECs. Stevens et al. [6
] isolated bovine PAECs using
Fig. 2 Overview of enzymatic isolation of pulmonary artery smooth muscle cells (PASMC ). (a ) Pulmonary arteries are removed from the lung and the adventitia is stripped using a combination of enzymatic digestion and forceps. (b ) The arterial ring is cut open and the endothe-lium is removed with gentle rubbing. (c ) The tissue is cut into small pieces for initial digestion by collagena. (d ) Following an overnight rest period in enzyme-free solution, new enzymes are added, the tissue is digested, and trituration releas isolated cells
488 C.V. Remillard et al.
an enzymatic dispersion technique first described for aortic endothelial cells by Coughlin et al. [7]. A similar approach has also been ud to isolate PAECs from lambs [8] (Fig. 3c ). Briefly, pulmonary arteries are discted into the lung paren-chyma, adventitia is removed as shown in Fig. 2a , and the lumen is washed with phosphate-buffered saline (PBS). The intercostal and distal branch ends are ligated or clamped, cut distally, and the lumen is filled with 0.1–0.25% collagena (type A from Roche or type 2 from Worthington) in Hank’s balanced salt solution. The digesting vesl (with ends occluded) is then immerd at 37°C. After approximately 20 min, the collagena solution is collected, the lumen is washed gently with culture medium, and the wash and colla-
gena solutions are pooled and centrifuged to pellet PAECs.
Fig. 3 Different techniques ud for isolation of pulmonary artery endothelial cells. Each procedure ultimately begins with isolated pul-monary arteries which are excid from the lung parenchyma. (a ) Cell scraping: arteries are inverted and expod endothelial cells are scraped off and plated. (b ) Enzymatic dissociation: arteries are minced, digested, and filtered to remove pulmonary artery smooth muscle cells and other cellular contaminants. Retrieved pulmonary artery endothelial cells are then purified for culture and/or experimentation. (c ) Isolated cells are filled with enzyme solution and ligated proximally and distally. Perfusate containing cells [including pulmonary artery smooth mu
scle cells, pulmonary artery endothelial cells, and fibroblasts] is filtered to isolate pulmonary artery endothelial cells. (d ) Magnetic beads: minced arteries are digested, then expod to magnetic beads coated with anti-IgG antibodies specific for pulmonary artery endothelial cells. Pulmonary artery endothelial cells with bound beads are iso-lated using a magnet, then resuspended and plated. (e ) Endothelial adhesion: arteries are cleaned and cut open, then laid in culture dishes with pulmonary artery endothelial cells facing down. Pulmonary artery endothelial cells gradually adhere to the culture surface, and attached pulmonary artery smooth muscle cells and adventitia are “peeled” off to leave adhered pulmonary artery endothelial cells, which are then plated and cultured
489 33 Isolation and Culture of Pulmonary Vascular Smooth Muscle and Endothelial Cells
The pellet containing endothelial cells is then resuspended in
culture medium and plated. For this technique, the culture
medium consists of a minimal esntial medium (d-valine)
supplemented with 20% calf rum, l-glutamine, penicillin,
and streptomycin.
We have isolated human PAECs via a modified enzymatic
technique first developed for mou lung PAEC isolation [9]
(Fig. 3d). In our procedure, tissues are minced and incubated
with M199 containing type 2 collagena and type 2 dispa
for approximately 1 h at 37°C. The digested material is then
filtered through a sterile 40-m m mesh and washed in 2% fetal
calf rum–M199. The cells are then incubated with
Dynabeads (Invitrogen), which are beads coated with sheep
anti-rat IgG and incubated with purified anti-platelet/endothelial
cell adhesion molecule (PECAM) or anti-CD31 monoclonal
antibody. Cells are expod to the antibody-coated beads for
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1 h at 4°C and bead-covered cells are captured by a Dynal
magnet. Once the supernatant containing unbound PAECs
and contaminating PASMCs or fibroblasts has been aspirated,
bound PAECs are resuspended in M199 and plated on glass
coverslips. As with human PASMCs, medium is changed
every other day until cells are confluent and ready for splitting
or experimentation. In our experience, the yield of purified
human PAECs via this technique exceeds 90%. A similar
procedure can also be ud to isolate PASMCs or fibroblasts,
using the appropriate cell-specific antibodies.
One final PAEC isolation technique involves neither enzy-
matic dispersion nor scraping PAECs off the arterial lumen,
and has been described for rat and bovine PAECs [10]
(Fig. 3e). The cleaned arteries are cut lengthwi and the
lumen is rind clean with PBS. The vesls are cut into 3–5-take out
mm2 pieces and placed lumen side down into a culture dish
for a few minutes until the tissues adhere to the dish surface.
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Tissues are then covered with 20% FBS–RPMI medium and
allowed to grow for 3 days in a normoxic (5% CO
2, 37°C)
incubator. Tissue pieces are then lifted carefully out of the medium and adherent PAECs are allowed to grow. After v-eral days, PAECs are trypsinized, diluted, and sparly plated (one or two cells per well in a 96-well dish) to lect for pure cobblestone-shaped PAECs. Selected cell populations ar
e then expanded, and cultured for experimentation.
4 C ell Subculture and Storage
averagely4.1 S ubculturing Cells
Becau large quantities of primary tissues can be difficult to obtain, subculturing of cells can be ud to amplify the sample size for future experimental u. Cultured cells should be passaged when they are subconfluent (approximately 70–80% confluent). The process itlf is quite straightforward, and the same procedure can be applied for many different cell types, including PASMCs and PAECs. Cultured cells are first washed with sterile HBSS or Dulbecco’s PBS to remove any dead cells or other cellular detritus. Washed cells are then treated with a trypsin–EDTA solution, which caus the cells to “ball up” and detach from the culture dish; full detachment typically takes less than 5 min, although this will vary according to temperature, cell confluence, cell type, and trypsin concentration. The trypsin exposure time should be kept as short as possibly as prolonged trypsin exposure may damage cell membranes and compromi cell viability. When most of the cells are floating freely, trypsin inhibitor or a trypsin neutralization solution is added to stop trypsiniza-tion. The cell suspension is then centrifuged and the pellet containing cells is resuspended
in warm medium for renewed plating. As with freshly isolated and plated cells, medium should be changed initially after 24 h and every 48 h thereaf-ter following each cell passaging.
The optimal cell density for passaging and plating varies with the size (i.e., surface area) of the plating dish. With commercially available cells, the supplier normally provides an estimate of the basal cell density. For tho using freshly dis-sociated cells, the cells must be counted to obtain an approxi-mation of the cell number. The optimal eding density is also dependent on the cell type. For PASMCs, we have determined cell proliferation is optimal at an initial eding density of approximately 3,500 cells/cm2. Cells eded at a higher initial density begin to proliferate earlier than tho eded at low density. Proliferation, if plotted as a function of time, is sig-moidal. In the initial lag pha, cell growth is minimal. However, within 2–3 days, cell growth increas exponen-tially during the logarithmic growth pha until confluence is attained, at which point growth is maximal and cells may begin to differentiate. In the prence of the required growth factors to stimulate cell proliferation, initial eding density and eding efficiency regulate the rate of cell proliferation.
4.2 C ell Storage
Cell storage in liquid nitrogen is the most efficient way to keep cells for long-term storage or for later u, especially tho cells originating from a rare tissue source. When the cultured cells are approximately 70–80% confluent, they are passaged as described earlier. However, instead of using medium, one resus-pends the final cell pellet in a freezing solution consisting of dimethyl sulfoxide (5–10%) and medium (FBS–DMEM for rat PASMCs, FBS–SMGM for human PASMCs, Endothelial Growth Medium (EGM) for PAECs). Cells in the freezing solu-tion should be aliquoted in cryogenic tubes, and gradually fro-zen until they can be placed in liquid nitrogen. Many core cell
