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,
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 9fold 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.
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
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
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.
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.
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 Dulbeccos 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
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 Dulbeccos 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
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.
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
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.
for this approach.
Why dont 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.
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
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.
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.
Childs GV, Naor Z, Hazum E, Tibolt
R., Westlund KN, Hancock MB 1983 Cytochemical characterization of
pituitary target cells for biotinylated gonadotropin releasing hormone.
Childs GV, Unabia G, Burke JA,
Marchetti C 1987 Secretion from corticotropes after avidin_fluorescein
stains for biotinylated ligands (CRF or AVP). Am. J. Physiol.
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
Childs GV, Lloyd JM, Unabia G,
Rougeau D 1988 Enrichment of corticotropes by counterflow centrifugation.
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.
Childs GV, Unabia, G, Miller, BT 1994
Cytochemical detection of GnRH binding sites on rat pituitary cells with
LH, FSH and GH antigens during diestrous upregulation. Endocrinology 134:
Childs GV, Unabia G, Wu
P 2000 Differential expression of growth hormone messenger ribonucleic
acid by somatotropes and gonadotropes in male and cycling female rats.
Endocrinology 141: 1560-1570
Childs GV, Unabia G 2001
The use of counterflow centrifugation to enrich populations of
somatotropes and gonadotropes. J Histochem Cytochem, in press
Kineman RD, Faught WJ, Frawley LS
1990 Bovine pituitary cells exhibit a unique form of somatotrope secretory
heterogeneity. Endocrinology 127: 2229-2235.
Moriarty GC 1973 Adenohypophysis:
Ultrastructural cytochemistry. A review. J. Histochem. Cytochem.
Villalobos C, Nunez L, Frawley LS,
Garcia-Sancho J, Sanchex A 1997 Multiresponsiveness of single anterior
pituitary cells to hypothalamic-releasing hormones: a cellular basis for
paradoxical secretion PNAS, 94 (25): 14132-14137.
Van Bael A, Denef C. 1996 Evidence
for a trophic action of the glycoprotein hormone alpha_subunit in rat
pituitary. J Neuroendocrinol. 8(2):99_102.
Vankelecom H, Denef C. 1997 Paracrine
communication in the anterior pituitary as studied in reaggregate cell
cultures. Microsc Res Tech. 39(2):150_6.
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