IN VITRO CELLULAR ~ DEVELOPMENTAL BIOLOGY Volume 25, Number 2, February 1989 �9 1989 Tissue Culture Association, Inc.

E N H A N C E M E N T OF P A N C R E A T I C I S L E T CELL M O N O L A Y E R G R O W T H BY E N D O T H E L I A L CELL M A T R I X AND INSULIN

ALBERTO HAYEK, ANA D. LOPEZ, AND GILLIAN M. BEATTIE

Lucy Thorne Whittier Children's Center, The Whittier Institute for Diabetes and Endocrinology, 9894 Genesee Avenue, La Jolla, California 92037

(Received 20 June 1988; accepted 3 October 1988~

SUMMARY

The roles of glucose and insulin in the promotion of DNA synthesis in pancreatic islet cell monolayers were assessed using a variety of in vitro conditions. Several substrates including collagen, poly-i- lysine, Matrigel, and the extracellular matrix produced by cultured bovine endothelial cells {BCEM~ were compared for their ability to promote monolayer growth. Islets grown on BCEM in combination with medium RPMI 1640 supplemented with 22.2 mM glucose or 10 gg/ml insulin gave the best results as determined by new DNA synthesis. The new-form monolayers were free of contaminating fibroblasts. These results suggest that insulin is critical to pancreatic islet growth when the cells are attached to biocompatible matrices.

Key words: pancreatic islets; monolayers; matrix; insulin; cell growth.

I NTRODUCTION

The observation that basal membranes produced by cultured ceils may be specific in their ability to enhance the growth of epithelial ceils ~6~ has been successfully applied to pancreatic islet cells in culture ~14,26~. Monolayers produced on extracellular matrices derived from cultured bovine corneal endothelial cells (BCEM~ exhibited increased cell proliferation (26} and the ability to remain functionally competent in response to different insulin secretagogues {14}.

We have compared the effects of several substrates in rat neonatal islet attachment and monolayer formation. Because both glucose (23,24~ and insulin (21} have been found to stimulate B-cell replication directly, we have examined their interactions with BCEM on islet cell monolayer formation and the promotion of DNA synthesis.

MATERIALS AND METHODS

Neonatal islet isolation. Islets were isolated from the pancreases of 3- to 5-d-old Lewis inbred rat pups by the method of Hellerstrom et al. (11) for fetal islets. Briefly, the pancreatic tissue was finely minced and digested for approximately 7 rain with coUagenase type 11 (Sigma, St. Louis, MOJ. After three washings in Hanks' balanced salt solution, the digest was plated into several 60-ram tissue culture dishes containing RPMI 1640 (GIBCO, Grand Island, NY} supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin}. The culture medium was changed daily and the cultures were continued for 5 to 7 d at 37 ~ C in a humidified atmosphere of 5% CO2 in air. On

the last day, the tissue was detached by gently blowing culture fluid with Pasteur pipettes against the bottom of the culture dishes; once floating freely all the islets were retrieved under direct vision with a stereomicroscope.

Preparation of monolayers from intact pancreatic islets. Handpicked neonatal islets were transferred to standard 60 )< 15-ram petri dishes in groups of 300 to 500 and allowed to free-float for at least 5 d in medium RPMI 1640, supplemented as before. The choice of RPMI 1640 and the concentration of FBS were based on previous reports showing this medium to be the optimum for preserving islet cell function and morphology (1,11,14}. After this period, groups of 5 to 10 islets were placed in each of 24-well polystyrene tissue culture treated plates (Multiwell, Beckton-Dickinson Labware, Lincoln Park, NJD. To facilitate islet attachment and monolayer formation, we used as substrates: collagen, poly-l-lysine, solubilized basement membrane produced by mouse sarcoma (Matrigel, Collaborative Research, Inc., Bed- ford, MA~, and BCEM. For some experiments, in addition to FBS, the culture medium RPMI 1640 was supplemen- ted with 22.2 mM glucose, 0.1 mM 3-isobutyl-l- methylxanthine (IBMX, Sigma Corp., St. Louis, MO}, or with 10 /~g/ml insulin (Crystalline porcine insulin was supplied by Dr. R. Chance, Lilly Research Laboratories, Indianapolis, IN}. We chose 10 ~g/ml of insulin because Rabinovitch et al. 121~ showed in serum-free medium, a dose-dependent effect on [3H}thymidine incorporation in B cells grown in monolayers. The collagen ICollagen Corp., Palo Alto, CA) and Matrigel were prepared according to the manufacturer's directions, and poly-l- lysine {Sigma Corp.~ was used at a concentration of 1 mg/ml. The BCEM muhiwell plates were precoated by

146

PANCREATIC ISLET CELL MONOLAYER GROWTH 147

TABLE 1

ISLET ATTACHMENT AND FORMATION OF MONOLAYERS WITH DIFFERENT SUBSTRATES AND MEDIA ADDITIONS ~

Substrate Type n Media Additions Attachment, % Monolayers, %

Collagen 5 35 0 Poly-L-lysine 2 100 0 Polystyrene 2 0.1 mM IBMX 90 0 Matrigel 2 22.2 mM glucose 100 0 BCEM 6 70 62 BCEM 2 22.2 mM glucose 86 80 BCEM 6 11.1 & 22.2 mM glucose ~ 100 100 BCEM 6 10 ~g/ml insulin 100 99

n = number of experiments. "There were 5 to 10 islets/well for a total of at least 100 islets for each experimental observation, islets were considered at-

tached when manual agitation failed to displace them from the matrix or substrate after 48 h. Percent monolayer formation refers to islets that formed monolayers after attachment after 5 d in culture.

bTwo days in 22.2 mM and 1 d in 11.1 mM glucose for the duration of the culture period.

the manufacturer (In Vitro International, Ann Arbor, MI). The percentages of attachment were determined at 48 h and of monolayer formation at 5 d. Microphoto- graphs were taken every 2 d until the monolayers began to retract or detach from the matrix.

Estimation of islet cell monolayer DNA synthesis. Only the monolayers grown on BCEM were used for these experiments, because the other substrates failed to induce monolayer formation. Under direct vision, 30 to 35 islets were plated on 15-ram wells coated with BCEM. The medium was R P M I 1640 plus 10% FBS and glucose to a final concentration of 22.2 raM. After 16 h, when all islets were attached, they were divided into three groups and kept in culture for the next 48 h under the following conditions: a) regular RPMI 1640 with 10% FBS, b) regular RPMI 1640 with 10% FBS supplemented to a final concentration of 22.2 mM glucose, and c) R P M I 1640 supplemented with insulin, 10 gg/ml, but without FBS. After this period, the medium was replaced in all wells by RPMI 1640 plus the FBS and incubated for 24 h; during the last 16 h, the cells were exposed to methyl- [3H]thymidine (sp. act. 58 Ci/mmol, ICN, Irvine, CA) at a concentration of 1 ~Ci/ml. After the [3H]thymidine pulse, the monolayers were trypsinized, washed twice with phosphate-saline buffer, and sonicated in distilled water. Thymidine incorporation in aliquots of the sonicates was determined by precipitation with 10% trichloroacetic acid and measurement of radioactivity in a liquid scintillation speetrophotometer. DNA content was determined by fluorometric assay of aliquots from the islet sonicates (13); the results were expressed as counts per minute/gg islet cell DNA.

Insulin release in response to glucose stimulation. Monolayers grown in the three different media, control, glucose supplemented, and insulin supplemented, were stimulated with glucose to measure the insulin released into medium. After a 2-d rest period in R P M I 1640 plus FBS they were washed twice in Kreb's Ringer bicarbon- ate buffer, pH 7.4, supplemented with 2 mg/ml of bovine serum albumin, and then were incubated for 1 h in the same medium with low (1.6 raM) or high (16.7 mM) glucose content. Insulin release into the buffer was measured by radioimmunoassay {I0).

Statistical significance was analyzed by Student's t tests for unpaired data.

RESULTS

Table I shows that attachment of islets to the different matrices was not by itself sufficient to promote monolayer formation. Attachment in the BCEM-coated dishes, however, especially when the medium was supplemented with glucose or insulin, led invariably to the uniform development of monolayers as examined under phase contrast microscopy. Examples of islets attached after 16 h under the above-described conditions, just before the initiation of monolayer formation, are shown in Fig. 1. Figure 2 depicts the marked enhancement of monolayer formation when influenced by supplements of glucose or insulin in the media. This enhancement was detectable by 24 h, clearly evident at 48 h, and maximum at 5 d. By 10 d the monolayers exhibited areas of retraction and cell detachment.

Although we could not visually quantitate the differ- ences in monolayer proliferation between the glucose- and the insulin-supplemented media, Fig. 3 shows that indeed insulin is a more effective promoter of DNA synthesis than glucose. The figure also shows that both media supplements, glucose and insulin, significantly

FIG. I. Phase contrast photomicrograph of three neonatal islets attached to BCEM at 16 h of plating and just before beginning of monolayer formation. X 100.

148 HAYEK ET AL.

FIG. 2. Phase contrast photomicrographs ()<200) at 48 (A, C, E)and 120 h (B, D, F)of pancreatic islet cell monolayers, A and B: controls; C and D: 22.2 M glucose supplementation; E and F: 10 /~g/ml insulin supplementation.

enhance DNA formation when the monolayers are grown in BCEM.

Table 2 shows that after 5 d in culture the monolayers grown in control or high-glucose-supplemented medium significantly released insulin in response to glucose stimulation. In contrast , the response to glucose s t imula t ion was not s ign i f ican t in the insul in- supplemented medium. Of note is the high basal insulin secretory rate measured in glucose-supplemented or insulin-supplemented medium, both significantly higher than the rate in the control medium (P = 0.001).

D ISCUSSION

Data presented in this report indicate that formation and growth of pancreatic cell monolayers are greatly enhanced when whole neonatal islets are placed on culture plates using BCEM as substrate and medium supplemented with glucose or insulin. An important

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FIO. 3. Effects of high glucose and insulin supplementation on DNA synthesis in pancreatic islet cell monolayers at Day 5 of culture.

PANCREATIC ISLET CELL MONOLAYER GROWTH ] 49

TABLE 2

GLUCOSE-STIMULATED INSULIN RELEASE {uU PER ISLET PER H) FROM MONOLAYERS GROWN ON BCEM AND GLUCOSE- OR INSULIN-SUPPLEMENTED MEDIUM ~

Glucose

Group 1.6 raM 16.7 mM P

Control medium 0.82 -4- 0.35 6.32 __. 1.28 <0.001 22.2 mM Glucose 4.38 ___ 1.08 6.13 -4- 0.55 <0.02 10t~g/ml Insulin 5.48 + 0.95 5.93 -4- 0.56 N.S.

"Each well contained 10 islets at the beginning of monolayer for- mation. Insulin release was carried out on Day 5 after attachment. Mean "4" SD.

advantage of this method is the virtual elimination of contaminating flbroblasts during the 10-d culture period, perhaps a reflection d the greater purity of the islet preparation which was achieved by the 5-d period of free-floating before transfer to the BCEM-coated dishes. Growth of flbroblasts has been a serious obstacle to the growth of pancreatic islet cell monolayers, as documen- ted by the multiplicity of attempts by means of media additions or subtractions to inhibit their proliferation (20,22). It is clear that in these experiments such effects cannot be attributed to inhibition by insulin or high glucose, because fibroblast proliferation has been reported to be enhanced by insulin (2) and unaffected by high glucose content in the media (3).

Recently, Kaiser et al. (14) were also able to grow islet monolayers using fragmented adult islets on BCEM. Fihrohlast contamination in their system was present and treated with thimerosal. In our own preliminary observations, digestion of the whole neonatal islets immediately after the initial 5-d culture period also led to extensive fibroblast contamination (not shown). There- fore, the period of free-floating of the whole islet as used in these experiments seems to deter the growth of contaminating fibroblasts.

Ohgawara et al. (19) first noted the inhibition of fibroblast growth during monolayer formation when whole adult rat islets were cultured in medium containing high glucose and IBMX. We however, using neonatal rat islets as shown in Table 1, were not able to induce production of monolayer formation in polystyrene tissue culture-treated dishes with IBMX-supplemented medium alone. Perhaps under these conditions age modifies the in vitro behavior of cells.

We chose neonatal islets because of ease of isolation, high yield from the neonatal dorsal pancreatic region, absence of exoerine tissue contamination (8), and the fact that they undergo the same in vitro neoformation process that Hellerstrom et al. (11) first reported for fetal rat islets. This process of in vitro neoformation has been recently well-characterized both morphologically and functionally (17,18).

The finding that BCEM, as compared to other substrates, favors the development of pancreatic islet cell monolayers is in accordance with previous reports demonstrating high growth rates for other epithelial cells of fetal origin grown on similar matrices (4). A previous

report using islet cell suspensions on BCEM-coated dishes also showed increased DNA synthesis when compared to cells grown on plastic (26L

It has been well demonstrated that glucose stimulates B-cell replication in vitro (12,23,24), and that rates of DNA synthesis a r e higher in fetal than in adult free-floating islets (24). Therefore, our results of the proliferative capacity of islet cells obtained from neonatal islets parallel those previously obtained with fetal islets. Also, high glucose has been shown to promote monolayer formation in cultured whole pancreatic islets (19L

In contrast to the favorable effects of glucose on islet cell growth in vitro, the situation in vivo is quite different. We and others have reported that transplanted islets (8) or islets in their native environment such as the fetal pancreatic islets in pregnant diabetic rats (5,7,25) grow and function poorly in a hyperglycemic milieu. In the case d the fetal islets from diabetic pregnancies, administration of insulin leads to morphologic (5,25} and functional (7) recovery. Equally successful for the outcome of the transplanted neonatal islet is the administration of exogenous insulin to the recipient animals in amounts sufficient to restore normoglycemia (9). These data suggest that islet cell replication is, if anything, favored by glucose-supplemented media and that replacement of the glucose by insulin is an even more potent stimulus for islet cell replications. As shown by the [3H]thymidine data in Fig. 3, it may well be that the apparent discrepancy between in vivo and in vitro glucose effects is due to the exposure to different insulin concentrations at the islet cell level.

Not surprisingly, the observed elevated basal insulin production rates (Table 2) are probably a reflection, at least in part, of the high B-cell replication activity induced by insulin. Also, the higher rates seen in the high glucose and insulin-supplemented media may reflect, at least in part, the greater number of cells as shown by the data in Fig. 3. In this respect, King et al. (15) have shown that B-cell replication and insulin release, under special circumstances, may be independent of each other,

Rahinovitch et al. (20) first reported a role for insulin in stimulating islet B-cell replication in neonatal rat pancreatic monolayer cultures in medium supplemented with ]6.7 mM glucose and 10/~g/ml insulin. Because they found that the dfect of insulin diminished in the presence of 0.1% serum, we omitted serum from our experiments. It well may be that the recently demonstra- ted inhibition by serum of the proliferative potential of mouse embryo cells (16) also applies to perinatal endocrine pancreatic cells when they are grown in culture. Rahinovitch et al. (20) also presented evidence that insulin-stimulated [3H]thymidine incorporation in B cells was mediated by a receptor for insuliulike growth factors rather than by the "classic" insulin receptors. This finding might explain the high concentration of insulin needed to demonstrate its mitogenic effect.

In summary, we have presented data to suggest that the combination of a matrix made of BCEM and medium containing high insulin concentrations in the absence of serum offers excellent growth conditions for pancreatic

150 HAYEK ET AL.

endocrine cell monolayers started with whole neonatal rat islets.

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The authors are grateful to Mrs. Eileen VanderLaan for her editorial help and to Elizabeth E x u m for the preparat ion of this manuscript. This work was supported in part by grant 187159 from the Juveni le Diabetes Foundat ion and B R S G RR05876 from the Nat ional Insti tutes of Health, Bethesda, M D .