Pancreatic islets were obtained from guinea pig pancreas by the collagenase method and kept alive in tissue culture prior to further studies. Pancreas cell morphology was studied by standard histochemical techniques using light microscopy. Preparative vertical electrophoresis-levitation of dispersed fetal guinea pig pancreas cells was conducted in phosphate buffer containing a heavy water (D20) gradient which does not cause clumping of cells or alter the osmolarity of the buffers. The faster migrating fractions tended to be enriched in beta-cell content. Alpha and delta cells were found to some degree in most fractions. A histogram showing the cell count distribution is included

Vanoss, C. J.

English

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https://ntrs.nasa.gov/search.jsp?R=19780019794 2020-03-22T02:36:03+00:00Z
F i
STATE UNIVERSITY OF NEW YORK AT BUFFALO
•
FINAL REPORT
	 HASM c 
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Date:	 6/5/78
Subject: ASSESSMFNT OF PANCKEAS CELLS NAS 9-15365
Part 1.	 Dispersion, culture, viability and electrophoretic mobility.
Part 2.
	
	 Temperature sensitivity, insulin content and/or production
and cellular morphology.
Part 3.
	
	 Ground-based simulation of cell fractionation by preparative
electrophoresis.
References
Figures
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Part 1.	 Dispersion, culture, viability and electrophoretic mobility.
Adult guinea pig pancreas
Pancreatic islets have been obtained routinely from adult guinea
pig pancreas, using the collagenase method of Goldman and Colle (1). These pancreas
are first cleaned of fat, connective tissue and large vessels, washed in Hanks' Balanced
Salt Solution (HBSS) and then minced to about 4 mm  pieces. These are shaken gently
at 37°C for 60 minutes, in a small tube containing 2.5 ml of HBSS anc collagenase (10
mg/ml). After digestion has taken place, the suspension is centrifuged at 30 x g and
the supernatant (containing collagenase and debris)'is discarded. The residue is washed
3 times with HBSS and then suspended in 3 ml Ficoll-Hypaque (density = 1.080). This
is overlaid with HBSS and then centrifuged for 5 minutes at 800 x g. The islets are
collected at the in' :rface and separated from cellular debris and non-islet cells (which
sediment to the bottom) (2). About 150-300 islets per pancreas normally are recovered.
These cells/islets are identified according to established histologic procedures (Part 2)
and subsequently kept alive in tissue culture prior to further studies.
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The islets are then explonted into 60 mm diameter Petri dishes con-
taining 15 ml CMRL 1066 (+ glutamine and 10% guinea pig serum) and placed in a
37°C CO2 incubator (1). The medium is changed every four days. Viability of
cells waintained in this way is at least 90% as judged by the trypan blue exclusion
test. (Briefly, a drop of islet-containing suspension is mixed with a drop of 0.4%
trypan blue dissolved in HBSS and the resultant mixture is evaluated immediately by
light microscopy). Those cells/islets that take up the dye are judged dead and those
that exclude the dye are judged living.
Electrophoretic mobility of cells obtained by collagenase-trypsin
dispersion (3) of adult guinea pig pancreas is measured by analytical cell microelectro-
phoresis (4). A mobility profile (histogram) results, with values ranging from — 1.30
to — 2.78 pm/sec/V/cm with a mean of —1.99 pni/sec/V/cm (see Fig. 1, page 8).
(The buffer used in this case was the one that earlier showed the best resolution in
preparative electrophoretic levitation (5): ionic strength 0.04, pH 7.3 phosphate
buffer).
Fetal guinea pig pancreas
Cell suspensions have been obtained from fetal guinea pig pancreas
in much the some manner as from adult guinea pigs, with a few differences in method-
ology. The density gradient step for islet separation is omitted, and a second col-
lagenase digestion, identical to the first is used to yield a suspension suitable for culture.
The cells are kept alive in the same manner as the adult material.
Viability of these cells, as judged by the trypan blue exclusion test, is about 75%.
A mobility profile (histogram), constructed from analytical cell
microelectrophoresis data is seen in Figure 2, page 9 . Mobility values ranged from
— 1.37 to —2.09 pm/sec/V/cm, with a mean — 1.76 pm/sec/V/cm. A phosphate
buffer, pH 7.3, ionic strength 0.04, was also used in these measurements.
-2-
F4
i '1 41 7
Part 2.	 Temperature sensitivity (a), Insulin content and/or production (b), and
cellular morphology (c).
a)	 A portion of a collagenose digest of adult guinea pig pancreas
was aliquotted into several small tubes after suspension in "freezing" buffer A-2
(developed in an earlier NASA-sponsored project and reported previously (5). These
were frozen by means of liquid N2 in a controlled rate freezer at a rate of —1°C/
minute to —58°C and then conserved in liquid N2 (6). Several tubes were removed
from the liquid after about six months and thawed at 37°C. Viability was assessed by
the trypon blue exclusion method. The cells were 70-80% viable as judged by this
method.
(b) Insulin content and/or production was not measured because
the required materials (including a large equipment item) were not available during
the time frame covered by the contract.
(c) The study of pancreas cell morphology has been pursued
exclusively by standard histochemical techniques using light microscopy. A repre-
sentative collection of cell types and stains may be seen in Figures 3 and 4,
page 10.
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Part 3.	 Ground-based simulation of cell fraction by preparative electro-
phoresis.
Preparative vertical electrophoresis-levitation of dispersed fetal
guinea pig pancreas cells was conducted in phosphate buffer containing a heavy water
(1)20) gradient. I A representative cell distribution obtained by this means is shown
In Figure 5, page ll.. The faster migrating fractions tended to be enriched in P-cell
content. Alpha and delta cells were found to some degree in most fractions.
The electrophoresis procedure parallels one previously published
by us in which the separation of populations of human lymphocytes is described (5).
A description of the new method, using a D2 0 gradient follows.
PREPARATIVE ELECTROPHORESIS OF CELLS BY ELECTROPHORETIC
LEVITATION IN A D20 GRADIENT
MATERIALS
Cells.	 Guinea pig pancreas cells were obtained by collagenase
digestion of fetal guinea pig pancreas (1).
Buffers. Phosphate buffers of low ionic strength (p = 0.04, pH 7.3)
were rrepared in distilled water or heavy water (D 20; Aldrich Chemical Co.,
Milwaukee, WI), consisting of 22.5 mg anhydrous No 2HPO4, 22.5 mg NaCI,
10 mg No2 EDTA and 600 mg glucose for a total volume of 20 ml.
1 D2 0 based buffers for increased density, or density gradients offer considerable
advantages over sucrose, Ficoll or other polymers, in that they neither cause clump-
ing of cells nor alter the osmolority of the buffers.
-4-
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Electrophoresis opEaratus. A vertical starch gel electrophoresis frame
• (Buchler, Fort Lee, NJ) was used to support a cylindrical polystyrene electro-
phoresis tube, 25 cm in length and 0.75 cm 1. D., prepared from a disposable pipet
(Falcon Plastics, Los Angeles, CA). This tube requires a water jacket of 4.4 cm 1. D.
for adequate cooling. A refrigerated water bath (LoTemtrol 154, Precision Scientific,
Chicago, IL) was used for circulating cooled water through the water jacket. Wicks
for the electrophoresis tubes consist of plugs of 2% agarose (Fisher Scientific,
Rochester, NY), (dissolved in the some buffer as used for the electrophoresis) at the
bottom and a filter paper bridge to the anode chamber at the top.
METHODS
Electrophoresis. After having been provided with an agarose plug at
the bottom, the electrophoresis tube is connected to the cooling water bath and chilled
to 4°C. At this point about 0.5 ml of buffer ( p = 1.124) prepared in D 2 0 is pipetted
above the agarose plug. Then, 7 x 10 6
 pancreas cells,suspended in 0.2 - 0.5 ml of a
mixture of equal parts D20/H2 0 buffer is layered on the D2 0 buffer cushion. The
rest of the tube is filled using 1 ml layers containing decreasing amounts of D20.
This gives a density gradient of 1.072 at the point of sample insertion to 1.020 at
the top (anode) of the electrophoresis tube. The wicks are connected immediately
and an electric field of 5 V/cm is applied vertically across the column with the anode
on top for 1 hour at 4°C. (The current may rise to about 8 mA, but care should be
taken not to allow the current level to exceed that value, otherwise a convective
plume of cells will form).
i -action collection.	 At the end of the electrophoresis a diffuse zone
of cells is visible. Samples of 1 ml each are taken from the top down by means of a
9 inch siliconized Pasteur pipet. Sampling is facilitated by the use of tF-e pipet
graduations on the electrophoresis tube. After a cell count` of each fraction, the
cells are transferred to CMRL-1066 containing 10% fetal calf serum and allowed to
rest before being stained for evaluation of cell type.
-S
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RESULTS
Twelve fractions were recovered containing a total of almost 6 x 106
cells. A histogram showing the cell count distribution is given in Figure 5. p-cells
were concentrated most heavily in the anodal fraction. Fraction 10 - 12 did not
contain alpha or delta cells, but did contain beta cells and some endocrine cells.
-6-
-7T--TT }
REFERENCES
1. Goldman, H. and Coll*, E., Science 192, 1014 (1976).
2. Tsai, C-M., et al., Transplantation 22, 201 (1976).
3. Lambert, A. E., at al., Endocrinology 90, 239 (1972).
4. van Cass, C.J., et al., Analytical Biochemistry 60, 242 (1974).
5. Gillman, C.F., et al., Preparative Biochemistry 4, 457 (1974).
6. van Cass, C. J. ' J. Reticuloendothelial Soc. (in the press).
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Figure 3. Pnotonicroarapii of as adult guiiwa pi- pancrc;,s tissu':
section s : iow.ng a large islet, stained by Got„ori's al4!u ,iydc-fuc.., :ia
metaod. iiote to dare:-stained granules in the beta cells. Tae lar; ;e ciar.;
area at tiie uottom is a staining artifact. Ma-niiicatio.: is 1OUx.
Figure 4 . Paotonicro;;r:.pa of au adult j;uinea Pi;, islet ( ur. sCF.:...	 i ,
isolated. after collageuase treatr.u.iL of miztcec: Pa.icrans, J c—. riiu,n..Lia..
on Ficoll-,iy?ariuc. density - 1.J.00.	 lie La cells ..re uoL a	 .ia ... ...:,.ale _..
ua^;L.► i.ied i51cCS. '•'agnificaLioa is 100x.
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1	 2 3 4 5 6 7 8 9 10 11 12
Frocti on	 Number
Figure S. Preparative electrophoresis histogram of 7 x 10 6 guinea pig pcncreass
cells. Electrophoresis in pil 7.3, 4-0.04 phosphate buffer. Electric
field strength: S V/cm. Duration 1 hour. Buffer density 1.072 , fraction
1 ) to 1.020 ( fraction 12 ).
- 11 -


Assessment of pancreas cells

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