21, 296-302 (1984)
Investigations into the Structure Rat Islets of Langerhans
MEZZOGIORNO of Anatomy,
of Naples, Naples 80139, Italy
Fetal rat pancreases, cultured for 8 days in RPM1 1640, were successively frozen to -196°C. The samples, defrosted at different intervals (0, 5, I, 2, 7, and 15 days). were examined by TEM and SEM. The effects of culture, various cooling times, warming rates, thawing procedures, dimethyl sulfoxide concentration. and ultrastructural features of cellular elements were analyzed.
FeTtal pancreatic tissue is useful in the remaining tissue was placed in 5 ml of RPM1 1640 (Flow Laboratories) contained in a 5achievement of experimental diabetes (6, 7, cm petri dish. The culture medium was sup10, 11, 14). The advantage of using fetal plemented with 10% fetal calf serum, 2 mg/ pancreatic tissue, rather than adult tissue, ml cefuroxine-sodic, 1 mg/ml tobramicine is that the islets in the fetal pancreatic tissue sulfate, and 1% NaHCOX. The remaining are capable of division both in vivo and in tissue was cultured at 37°C for 18 days in vitro. In addition, fetal pancreatic tissue ap95% air, 5% CO, (v-v) at 100% humidity. pears to be more resistant to the rigors of Each day the nonfloating residual tissue was freeze-thaw injury. In this study, pancreases drawn into a sterile 5-ml syringe (without from full-term fetal rats were partially dia needle) and transfered into fresh culture gested, cultured for 8 days, and then either medium. By the end of the eighth day, little examined directly or after freezing and exocrine pancreatic tissue remained. thawing by transmission and scanning elecThese culture conditions were established tron microscopy. to facilitate the destruction of the acinar MATERIALS AND METHODS tissue without damage to the endocrine tissue. An average of 250 morphologically inPancreases were isolated from fetal Wistar rats (gestational age 19 days). The sur- tact islets were isolated from each culture under stereo microscope. Approximately gically removed pancreases were immersed in sterile Hanks’ solution, minced into pieces 25 islets were placed in each of 10 test tubes containing 10% dimethyl sulfoxide approximately 0.5-l mm on a side, and then (Me,SO). The islets were incubated at 22°C digested in collagenase (Sigma Type 5, Lot for 20 min and then the temperature was C-9263, or Boehringer-Mannheim, Lot lowered to 4°C and the islets were held at 1149430). Three milligrams of a collagenase was used for each pancreas and usually 12 this temperature for 1 hr. The freezing technique used was similar pancreases were used per isolation. The tisto that currently in use in our laboratory sue was digested for 15 min in a shaking for the cryopreservation of other fetal mawaterbath equilibrated at 37°C and agitated terial (8, 9). A slow freezing protocol was at 100 cycles per minute. After digestion, used which involved cooling the cell susthe residual enzymatic activity was removed by washing in cold Hanks’ solution, and the pension at 0.25”C/min to -7”C, holding at this temperature for 20 min, and then resuming cooling at 0.25”C/min to -196°C. Received June 6, 1983; accepted July 21, 1983. 296 001 l-2240/84 $3.00 Copyright 0 1984 by Academic Pros, 1~. All rights of reproduction in any form reserved.
MORPHOLOGY OF FROZEN-THAWED
RAT ISLETS OF LANGERHANS
TABLE I Insulin Release from Fetal Rat Pancreatic Islets Immediately after Isolation, after 8 Days of Culture, and after Culture and Various Storage Times Glucose concentration in the culture medium (g/liter)
41.3 + 6.5 23.7 + 5.6
65.1 k 3.8 46.9 + 4.1
38.6 k 5.1 26.3 f 6.0
After 8 days of culture and days of storage in liquid Nz
The freezing rate was precisely controlled by the use of a Cryo-diffusion PN2 temperature-controlling electronic computer (Cryo-diffusion, France). Samples were stored in liquid nitrogen for 0.5, 1, 2, 7, or 15 days and the samples were warmed at 6 to 8”C/min until the samples reached -4°C. The samples were then transferred to a regulated waterbath at 37°C. The MeSO was progressively diluted with warm Hanks’ so-
35.4 I? 7.2 41.4 + 9.5 38.1 k 8.0 24.3 f 5.1 25.2 f 5.0 23.0 f 7.2
15 30.4 f 8.2 25.3 f 4.1
lution supplemented with 25 rnM Hepes [ 4(2-hydroxyethyl)- 1-piperazine ethane sulfonic acid], 0.025% fetal calf serum. The physiological competence of the islets was determined by testing the ability of the islets to release insulin in response to a glucose challenge. In order, the islets were incubated at 37°C for 1 hr in 1 ml of a challenge solution containing 4 g/liter of glucose, followed by a further incubation in a solution
FIG. 1. Scanning electron micrograph showing fetal rat islet cultured for 8 days in RPM1 1640. Note the presence of filopodia and zeiotic blebs. (x200).
FIG. 2. Scanning electron micrograph showing surface insular cells at higher magnification. The cells, organized in typical corde, appear in polygonal shapes. Note the numerous microvilli on their surface. (X520).
containing 0.04 g/liter of glucose. The supernatant of both the high and low glucose challenge were removed and kept at 20°C. Subsequently, the supernatants were defrosted and insulin levels were measured by radioimmunoassay, using a LKB minigamme spline (Mod. 1275). The rat insulin standards (Lot. 615 D63-12, mixture of insulin I and II, obtained from Dr. M. Tocca, Upjohn, Italy), were labeled with ‘251.Each supernatant was assayed in triplicate. The standard curve was established over the range of 0 to 200 PI-J/ml. All samples to be analyzed morphologically were fixed in 2.5% glutaraldehyde buffered in 0.1 M phosphate buffer (pH 7.2). Those samples to be viewed by scanning electron microscopy were postfixed in 1% osmium tetroxide, dehydrated in a graduated acetone series (5-10 min per wash),
and then equilibrated in several changes of acetone for a minimum of 30 min. The samples were criticalpoint dried directly from the acetone. The samples were mounted on a tube using conductive carbon cement (Neubauer Chemikalien), and subsequently sputtercoated with gold-palladium in a Polaron E 5000 sputtercoater and examined with an IS1 Super III scanning electron microscope. Those samples to be viewed by transmission electron microscopy were fixed in 3% glutaraldehyde and 4% paraformaldehyde in 0.67 it4 cacodylate buffer with 7.5% sucrose, pH 7.2. Specimens were washed in 0.1 M cacodylate buffer, postfixed in 1% osmium tetroxide, stained en bloc with uranil acetate, dehydrated, and embedded in Epon. Sections were cut with a glass knife on an LKB III ultramicrotome. Thin sections were stained
FIG. 3. Scanning electron micrograph showing two islets of a fetal rat (of different after frozen storage for 7 days in liquid nitrogen. (X70).
with lead citrate and many1 acetate and examined in a Zeiss EM 109 electron microscope. RESULTS
The results of the glucose challenge of fetal islets determined immediately after isolation, after 8 days of culture, and after 0.5, 1, 2, 7, and 15 days of storage in liquid nitrogen were summarized in Table 1. At the beginning of the culture period the islets become attached to the bottom of the petri dish; upon stereomicroscopic observation such islets were virtually free of exocrine cells and had a spherical or ellipsoid shape. Some islets were surrounded by fibroblastoid cells (Fig. 1). The frozenthawed islets examined by SEM showed few signs of morphological degeneration, except
in occasional large islets which sometimes showed the superficial cells with signs of necrosis or degeneration. The islets were arranged in a dense, epithelioid manner, and no acinar elements could be identified (Fig. 2). At high magnification, many of these peripheral cells appear to be polygonally shaped and possessvery distinct margins and extensive microvilli. The mean volume of the islet preparations was approximately 300 pm3 (Fig. 3). By transmission electron microscopy, the frozen-thawed islets were similar to those of the cultured material. Islets showed satisfactory ultrastructural preservation, with nuclei, mitochondria, and cellular membranes having a normal appearance. The endoplasmic reticulum ap-
FIG. 4. Electron micrograph showing the types of granulated insular cells in fetal pancreas of rat cultured and frozen for 15 days in liquid nitrogen. In the upper part of the figure the typical granulation of the p cell can be seen. In the lower portion, the other types, containing a variable number of granules. Stained with many1 acetate and counterstained with lead acetate. (X7000).
peared abundant in some cells but was poorly developed in others. The ,6 and cycells were easily recognized by the appearance of their secretory granules. However, in the frozen-thawed islets, the P-cell granules were rounder, with tighter vesicles. Comparisons of the ultrastructure of the cultured islets with cultured and frozen-thawed islets showed only a slightly higher frequency of damaged cells in the latter group. DISCUSSION
The morphological evidence presented here, in conjunction with the radioimmu-
noassay data concerning the release of insulin from islets frozen and thawed under similar conditions, lead us to believe that the cryopreservation regime used does not cause substantial irreversible damage to fetal rat islets of Langerhans. Our results are consistent with the relatively high survival rate reported by other authors for the preservation of fetal islets, through our freezing conditions are completely different. At this stage of embryonic development, there is a preponderance of ,8 over (Yand F cells. In the more mature islets, these /I cells resemble adult @cells. Specifically, the insulin-containing granular inclusions were
FIG. 5. Electron micrograph showing the granulation of pancreatic p cells of fetal rat at higher magnification. Stained with uranyl acetate and counterstained with lead acetate. (X17,000).
present, while less mature islets showed a huge number of vesicles lacking the typical insulin crystals (Figs. 4 and 5). In other islets, there were many bizarre cellular elements which defy cataloging and certainly represent an atypical morphology of the p cells. It is not clear from these studies whether such morphological perturbations are reversible. Large prolongations of the ,&cell plasma membrane were frequently seen. In general, the transmission and scanning electron micrographic evidence presented here confirm our previous light microscopic impressions that acceptable retention of morphological integrity can be preserved
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