The
use of counterflow centrifugation (centrifugal elutriation)
to enrich and purify corticotropes, gonadotropes, and somatotropes.
This work was done by Gwen V. Childs, Ph.D. Department of Anatomy and Neurobiology, University of Arkansas for Medical Sciences, Little Rock, AR and Department of Anatomy and Neurosciences, University of Texas Medical Branch. Galveston, TX.
Rationale for doing this work:
The anterior pituitary consists of different hormone-producing cells that can be differentiated cytochemically by the type of hormone produced (Childs, 1973) or their receptor population (Childs et al, 1983, 1984, 1994a, Villalobos, 1997). Pituitary cell populations are thus difficult to study because they are a part of a mixture of cell types. Neuroendocrine laboratories who want to study responses and functions of individual pituitary cell types have used tumor cell lines as models. Or, if they wanted to use primary pituitary cell types, they have sought ways to either separate the subsets and purify them (reviewed in Childs et al, 1988, 1992; Van Bael and Denef, 1996; Vandelecom and Denef, 1997), or provide identifying markers for the cells, especially in the living state (Childs et al, 1987;Childs and Burke, 1987; Kineman et al 1991).
This presentation will describe a method that produces a 9–fold enrichment of pituitary corticotropes (Childs et al, 1988), enriching them to 90% cells that bear adrenocorticotropin and endorphin antigens. The cells can be grown in culture and used for at least 7-10 days for electrophysiological studies. More recently, we have worked on techniques that enrich gonadotropes or somatotropes to provide cells for studies of proliferation or differentiated function. This presentation will describe the technology and its analysis.
The major principle behind the method is that pituitary cells enlarge in response to their specific releasing hormone. Therefore, we began by separating the cells into three fractions by centrifugal elutriation. This first step is diagrammed in the following Figure. Different flow rates are used to remove (elute) the different fractions; higher flow rates are able to elute larger cells. A gradual increase in flow rate elutes each fraction sequentially.
Cells in each of these fractions are stimulated for 3 hours with Gonadotropin releasing hormone (GnRH, to enrich gonadotropes) or Growth hormone releasing hormone (GHRH, to enrich somatotropes), or corticotropin releasing hormone (to enrich corticotropes)..
After stimulation, the cells are then re-eluted at higher flow rates to further separate the enlarged cells from their counterparts in each of the fractions. These new fractions are pooled to form the enriched cell population.
The protocols begin with the dispersion of pituitary cells by previously reported methods (Childs et al, 1988, 1994, 2000). Male or female Sprague Dawley rats are used for these protocols. The rats are acclimated 7-10 days before use. All care and use protocols have been approved by the committees at both University of Texas Medical Branch and University of Arkansas for Medical science. During the dispersion steps, care is taken to separate clumps so that single-cell preparations are obtained. Generally 4-6 rats are used for each separation. This produces 10-12 million pituitary cells and ultimately 1-1.5 million somatotropes or gonadotropes.
Step 1. Preparation of the Elutriator rotor.
The first step is to prepare the Beckman elutriator rotor (Childs et al, 1988). It is assembled with the Sanderson chamber, which is used because it allows work with small numbers of cells (range: 10 thousand to 10 million cells). The elutriator rotor is assembled in the centrifuge which is attached to a peristaltic pump and tubing that feeds fluid into the centrifuge rotor as it is spun. Initially, 500 mls of 70% ethanol is run through the rotor at about 10 mls/minute to provide sterilization. The centrifuge is not running during this treatment step. This is followed by a thorough wash (2 liters). Two liters of cold millipore filtered water containing gentamycin is run through the rotor at 40 mls/min. Care is taken to prevent and remove all bubbles during this phase. The water is in a sterile flask in an ice bath. The rotor is then pre-treated with Dulbeccos phosphate buffered saline also containing gentamyacin and bovine serum albumin (200 mls; 10 mls/min). During the last pretreatment, the centrifuge is run at 1960 rpm to check for leaks, bubbles, or other problems. It is opened after this test and all visible bubbles are removed from the system by hand spinning, squeezing the tubing, etc.
STEP 2. Loading the Cells
The centrifuge is then closed and run again at 1960 rpm and 0-4 C. The pituitary cells are loaded into a 10 ml syringe connected to the tubing running through the peristaltic pump. The loading process begins when the valve is opened and the cells loaded into the running centrifuge Loading flow rate is 8-10 mls/min. After the cells are loaded, the valve is shifted to load 50 mls of Dulbecco’s PBS at the same flow rate. Care is taken not to allow any bubbles to form or enter the system. This loading step allows the cells to settle into the Sanderson chamber with the largest cells at the bottom and layers of smaller cells at the top. It usually takes about 5 minutes. The loading fraction is collected because it contains some of the smallest pituitary cells.
Step 3. Recovery (elution) of each fraction.
Then, the flow rate is turned up to the next increment in order to collect larger cells in the next fraction. This flow rate is 15 mls/min and 50 mls are collected to make up Fraction 1. Fraction 2 (50 mls) is then collected at 25 mls/min and Fraction 3 (35 mls) is collected at 35 mls/min. The fractions are all collected in 50 ml tubes on ice. The third fraction is collected as the centrifuge is slowed to a stop which helps to push the largest cells out. However, there are still large cells remaining in the chamber and the tubing which must be collected. After the centrifuge is stopped, the rotor is removed and the fluid remaining in the tubing is emptied into the Fraction 3 tube (automatically when it is disconnected from the elutriator rotor). Then, the chamber is removed and a flexible plastic pipette is inserted into one port to remove the contents of the chamber. The chamber is then put back into the rotor and new Dulbecco’s PBS is pumped into the rotor, bubbles are removed and the system is checked for leaks. It is maintained in this state until after the cells are stimulated (for three hours).
The Loading fraction is pooled with Fraction 1 and contains the smallest cells. The chamber contents are pooled with Fraction 3 and include the largest cells. The cell fractions are spun down at 900 rpm and the pellets are resuspended in Dulbecco's Modified Eagle's media (high glucose-DME) including: 2.5 :g/500 ml HEPES buffer, 0.3% bovine serum albumin, 5 :g/ml insulin, 30 nM sodium selenite, 50 :g/ml transferrin, and 4.2 :g/ml fibronectin.
Step 4. Stimulation of fractions with secretagogues.
Then, stimulation with releasing hormones for 3 hours at 37 C is begun to enlarge either the corticotropes, gonadotropes or somatotropes. To produce enriched gonadotropes, the fractions are stimulated with with 1 nM Gonadotropin releasing hormone (GnRH); to produce enriched somatotropes, cells are stimulated with 1 nM Growth hormone releasing hormone (GHRH) and to stimulate enlargement of corticotropes, the cells are stimulated with 0.5 nM corticotropin releasing hormone (CRH). In the developmental studies, the cells in each fraction were measured before and after the 3 hour stimulation. The area measurements showed that a subset of pituitary cells enlarged as a result of the stimulation and cytochemical analyses demonstrated that the enlarged cells were targets of the specific releasing hormones.
Step 5. Re-elutriation of the stimulated fractions
After the stimulation period in either GnRH, CRH, or GHRH, each fraction is loaded at 10 mls/min (50 mls are collected). These fractions are eluted first at the original flow rate (Fr 1=15 mls/min; Fr 2=25 mls/min; Fr 3=35 mls/min). Then, the remaining, larger cells are eluted at a new flow rate. This collects the larger cells that emerge from each fraction and is 5-10 mls/min higher than that of the original. In the developmental phases of the study, samples were taken from the original fractions as well as the cells from the new enlarged cell fractions and immunolabeled for either growth hormone or gonadotropins.
Table 1 illustrates an experiment for the growth hormone protocol. GH cells were identified by immunolabeling as described in previous studies. The first two columns list the percentages of GH cells in each of the original fractions. The second two columns list the percentages of GH cells in the enlarged cell fractions derived from the re-elutriation of these fractions (at a higher flow rate) after stimulation. Note that all three re-eluted fractions contain significantly more GH cells. The average percentages of GH cells in the final fraction ranges from 91-93% cells with GH antigens.
These cells are then plated in 24 well trays on glass coverslips. They are grown in defined Dulbeccos media as described above. Table 2 shows that the cells maintain their enrichment after 2 days. Treatment with GHRH during the culture period did not alter the original enrichment.
There are two important differences in the enrichment protocol for gonadotropes, corticotropes, and somatotropes. First, the most obvious is the exposure to the two different secretagogues, as described above. Second is the flow rate used for re-elutriation. To collect the somatotropes, the enlarged cell fractions are eluted at 5 mls/min higher than the original fraction. However, to collect the larger gonadotropes or corticotropes, the new flow rates for each of the fractions are increased 10 mls/min. Thus, for gonadotropes or corticotropes, the original flow rate is 15 mls/min and the new flow rate used to separate the enlarged gonadotropes or corticotropes from their counterparts in the original fraction is 25 mls/min. Similarly, enlarged gonadotropes or corticotropes from fraction 2 are collected at 35 mls/min and those from fraction 3 are collected at 45 mls/min.
Rationale for this approach.
Why don’t we stimulate dispersed pituitary cells directly? Why do we start by separating them into fractions?
The reason for the two step elutriation process is that many of the smaller gonadotropes or somatotropes do not enlarge sufficiently to be separated from a population that already contains large cells. Most of the GH cells or gonadotropes enlarge by at least 10 :m2 . The elutriation protocol can separate cells that differ in area by 5-10 :m2. This means that a small cell only has to enlarge by that amount in order to be separated from its counterparts in the fraction containing other small cells. However, it might not become larger than the largest pituitary cells in the entire population. Therefore, we increase the chances for separation and collection by initially separating it with its counterparts. This increases the overall yield of each cell type.
How do we analyze the purified or enriched fractions?
The enriched fractions are analyzed by immunolabeling for their content of pituitary hormones. Table 3 shows the counts of different cell types in the gonadotrope-enriched fractions, comparing male and female rats. Percentages of all cell types are reduced significantly, except for the GH cell population. We have reported significant cellular overlap between GH and gonadotropin expression and these data reflect the presence of GH stores in gonadotropes, especially in female rats (Childs et al, 2000 ). A similar relationship is seen in the somatotrope populations; at least 15% of GH cells contain gonadotropin antigens.
Figure
1
Figure 2.
Figure 1 illustrates immunolabeling for LH and FSH in enriched gonadotropes showing the label in orange and black. The clear cells are unlabeled. Orange=FSH and black=LH.
Evaluation of the technology
The technique has both advantages and disadvantages. It does not provide a high yield of cells, suitable for protein purifications or biochemical assays. It might be possible to run this protocol with the Standard chamber which separates 50 million cells. Then, individual fractions which contain smaller numbers of cells could be re-eluted in the Sanderson chamber. The same principle of enlarging the cells by at least 5-10 :m2 could be applied. An important consideration must be the choice of a secretatogue that is reasonably specific. Thyrotropin releasing hormone, for example, would stimulate multiple cell types (including prolactin and thyroid stimulating hormone cells).
The major advantages of the technique is that it uses few animals and can be completed in one full day. It produces cell populations of around 1 million cells that are at least 90% gonadotropes or somatotropes. This makes it ideal for protocols that need isolated cells, or small cell populations. Examples of such studies include those that study cell proliferation, ion channel activity, or cellular communication. Potential uses for these cells might be to collect conditioned media and identify secreted products that might be used in autocrine or paracrine stimulation studies.
References Cited:
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Childs GV, Burke J 1987 Use of the reverse hemolytic plaque assay to study the regulation of anterior lobe ACTH secretion by CRF, AVP, A_II and glucocorticoids. Endocrinology 120:439_444.
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Childs GV, Unabia G, Lee BL, Rougeau D 1992 Heightened secretion by small and medium-sized luteinizing hormone (LH) gonadotropes late in the cycle suggests contributions to the LH surge or possible paracrine interactions, Endocrinology, 130: 345-352.
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Westlund KN, Wynn PJ, Chmielowiec S, Collins TJ, Childs GV 1984 Characterization of a potent biotin_conjugated CRF analog and the response of anterior pituitary corticotropes. Peptides 5:627_634.