Pancreatic Beta Cell Culture: Preparation of Purified Monolayers WILLIAM L. CHICK, ARTHUR A. LIKE, AND VILMA LAURIS Elliott P. Joslin Research Laboratory, Joslin Diabetes Foundation, Inc., The Harvard Medical School and The Peter Bent Brigham Hospital, Boston, Massachusetts 02215 ABSTRACT. Procedures were developed for preparing partially purified beta cell monolayer cultures. Neonatal rat pancreases were dissociated with a trypsin-collagenase solution. Beta cells were separated from denser acinar cells by centrifuging the initial suspension in a two layer discontinuous Ficoll gradient (20, 25%). Resultant cultures, highly enriched in beta cells, were further purified by incubation with cystine-free medium. This caused
necrosis of the majority of fibroblastoid cells within two days, while beta cells were considerably less affected. The resultant cultures contained an average of 72% beta cells compared to 10% in untreated control cultures. Insulin release by purified monolayers remained responsive to changes in the glucose concentration of the culture medium. (Endocrinology 96: 637, 1975)
M
ONOLAYER cultures containing pancreatic beta cells have been successfully employed to study insulin biosynthesis and release, beta cell replication, and beta cell morphology by both light and electron microscopy (1-7). Unfortunately despite recent advances in methodology for preparing such cultures from newborn rats, the beta cells still constitute only a small fraction of the total cell population. This obviously restricts their usefulness. Studies were therefore undertaken to develop means for preparing purified beta cell monolayer cultures from newborn rats. The method presented is based on Ficoll gradient centrifugation to separate endocrine from exocrine cells in the initial cell suspension (8,9), followed by incubation of the resulting cultures with a nutritionally deficient medium to eliminate the majority of residual extraneous cells. Potential applications for this method are discussed. Materials and Methods Preparation of monolayers Cell suspensions were prepared by dissociating pancreases from two to three day old rats (C-D strain, Charles River) with trypsin and collagenase as previously described (4,5). An average of 70 pancreases were used in each experiment: The cell count was adjusted to Received September 23, 1974.
7.5 x 105 viable cells per ml, and 5 ml removed to prepare a control culture. The remainder of the suspension (approximately 180 ml) was centrifuged at 150 x g for 5 min and the cell pellet resuspended in 4 ml of an ice cold solution of 25% (wt/vol) dialyzed Ficoll (9) in growth medium. Growth medium was TCM 199 containing 10% fetal calf serum, 400 U of sodium penicillin per ml, and 300 rng of glucose per 100 ml unless specified otherwise. The cell suspension was overlaid with an additional 4 ml of 20% Ficoll in growth medium and centrifuged for 10 min at 1000 x g. Preliminary experiments indicated that the portion of the gradient from the 20-25% interface to just above the cell pellet was highly enriched in beta cells, while acinar cells remained in the pellet. This fraction enriched in beta cells was removed, diluted with growth medium and centrifuged to remove the Ficoll. The final pellet was suspended in 10 ml of growth medium and divided equally between two cultures. Sixty millimeter petri dishes were used for all cultures. Incubation was at 37 C in a humidified atmosphere of 95% air: 5% CO2 as previously reported (4,5). The primary cell suspension was decanted into new dishes after 14-16 h to eliminate large numbers of fibroblastoid cells which attached to the original dishes (3-5). The following day the control and one of the Ficoll-treated cultures were washed with growth medium and reincubated. The other Ficoll-treated culture was washed and reincubated with cystine-free Eagle's Minimal Essential Medium (Grand Island Biological Company Select-amine Kit) containing 10% dialyzed fetal calf serum, 300 mg of glucose per 100 ml, and
637
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Endo • 1975 Vol 96 • No 3
CHICK, LIKE AND LAURIS
638
400 U of penicillin per ml. Incubation with the cystine-free medium led to rapid necrosis of the overwhelming majority of residual fibroblastoid cells while significant numbers of endocrine cells survived this treatment. After 2 days, all three types of cultures were again washed and reincubated with growth medium for two additional days. Finally, during the last 2 days of the experiment the glucose concentration in the growth medium was lowered to 100 mg/100 ml to permit regranulation of the beta cells and thus facilitate identification following staining with aldehyde-thionin (Fig. 1). In addition, in three experiments the effects of longer-term incubation were evaluated by maintaining cultures for a total of 3 weeks rather than 8 days. The protocol followed was similar to that already described with the exception that the glucose concentration in the medium was lowered from 300 to 100 mg/100 ml during the 2 days prior to fixation and staining rather than during days 6 to 8.
face of each culture dish. Cells in each field were assigned to one of three categories depending upon their shape and staining characteristics: 1. Fibroblastoid; 2. Epithelioid, aldehyde-thionin positive (beta cells); 3. Epithelioid, aldehyde-thionin negative.
Results
Morphology Gradient separation. Four layer Ficoll gradients (25%, 23%, 2Q%, 11%) similar to those used to isolate islets from adult rats were initially employed to develop the two layer gradient used in these experiments (8,9). Using this simpler gradient, the
majority of cells remained at the bottom of the 25% Ficoll layer. The predominant cell type in this pellet, as evaluated by phase contrast microscopy (4,5,7), consisted of acinar cells. Cultures prepared from this portion of the gradient contained some beta Insulin release cells, as evidenced by aldehyde-thionin Aliquots of medium (Fig. 1) were assayed for immunoreactive insulin (IRI) content as previ- staining. These presumably had formed ously described, using purified rat insulin stand- aggregates with the denser acinar cells and sedimented in the gradient with them (7). ards (4,5). Cultures prepared from the upper portion of the 20% Ficoll layer contained relatively Light microscopy and morphometric analysis few cells. There were rare aldehyde-thionin Living cultures were examined periodically positive (beta) cells in addition to fibroblasby phase contrast microscopy. After 8 days of toid appearing cells. The portion of the incubation they were fixed and stained with gradient from the 20-25% interface to just aldehyde-thionin for identification of beta cells above the pellet at the bottom of the (3-5). Because of rapid fading of stained fibroblastoid cells, morphometric studies were per- gradient yielded cultures enriched in formed immediately following staining. A aldehyde-thionin positive (beta) cells, but minimum of 1000 cells were counted in ran- also containing significant numbers of domly selected fields distributed over the sur- fibroblastoid cells. The majority of these fibroblastoid cells attached to the original Plonl Sove Medium Sove Medium dishes during the initial 14-16 h of incubaDecora for IRI forlRI Cultures and were therefore discarded when tion I I the cell suspension was decanted into new Doys : 0 e dishes (3-5). Medium : 199 199 199 199or cystine-free MEM
Glucose KX>mg/)0Onl (Regroiiulotion F^riod)
FIG. 1. Protocol followed in preparing and studying control and purified cultures. The glucose concentration in the culture medium was 300 mg/100 ml except during days 6-8 when this was lowered to 100 mg/ 100 ml to permit beta cell regranulation prior to staining.
Cystine-free medium. The use of cystinefree medium was based on our observation that incubation of rat pancreatic monolayer cultures with 100% fetal calf serum resulted in obvious necrosis of the majority of fibroblastoid cells within 1-2 days. Under
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PANCREATIC BETA CELL CULTURE these same conditions the beta cells in these cultures were affected to a considerably lesser extent. Subsequent amino acid analysis of the fetal calf serum used in these experiments revealed that of the amino acids reported to be essential for most mammalian cells in culture (10) all were present in adequate amounts with the exception of cystine. The half-cystine content of the fetal calf serum was only 2 /x,mol per liter. The necrosis of fibroblastoid cells in the cultures observed under phase contrast microscopy could be prevented by the addition of cystine (200 /xmol per liter) to the fetal calf serum (11). Finally, the effects of 100% fetal calf serum without added cystine were duplicated by the use of cystine-free Eagle's Minimal Essential Medium (MEM). As anticipated (6), complete MEM exerted no discernible morphologic effects. . Although the cultures in the majority of experiments included in this report were incubated for a total of 2 days with cystinefree MEM, smaller numbers of experiments were also conducted in which incubation was continued for a total of 5 days. Although fibroblastoid cells were almost totally eliminated after this longer incubation with the nutritionally deficient medium, the numbers of endocrine cells surviving were very markedly reduced. From these experiments it was concluded that a 2-3-day incubation with the cystinefree medium was optimal in terms of preserving significant numbers of beta cells while still achieving relatively high purity through elimination of extraneous cell types. Although cystine is an essential amino acid for most mammalian cells in culture, it is not an essential amino acid for the whole animal, since it is synthesized in the liver from the essential amino acid methionine and the nonessential amino acid L-serine (12). In view of the possibility that similar conversion pathways were also present to a limited extent in beta cells, cultures were incubated in cystine-free MEM containing ten-fold increased levels
639
of L-methionine and to which the nonessential amino acid L-serine was also added (42 mg per liter). Under phase contrast, cultures incubated with this medium were indistinguishable from those incubated in cystine-free medium without the added amino acids. Since tyrosine is a second amino acid essential for most mammalian cells in culture, but not for the whole animal (10), the effects of tyrosine-free MEM were also evaluated. Interestingly, after 3 days of incubation, no obvious effects could be discerned by phase microscopy on either fibroblastoid or epithelioid cells. The construction of these deficient media was facilitated by the availability commercially of a kit designed to permit deletion of specific amino acids in MEM. Qualitative evaluation of the three types of cultures. As previously reported (4), control cultures contained numerous scattered col-' onies of epithelioid cells surrounded by a dense network of fibroblastoid cells. The majority of these epithelioid cells contained aldehyde-thionin positive granules (Fig. 2A). In contrast, cultures prepared by Ficoll gradient separation contained considerably greater numbers of these epithelioid colonies. In addition, the colonies were noticeably larger in size than in the control group. The majority of these epithelioid cells were also aldehydethionin positive (Fig. 2B). Although fibroblastoid cells occupied the spaces between the epithelioid colonies, their numbers were limited by the large amount of space taken up by the latter. Finally, cultures prepared by Ficoll gradient separation and then treated with cystine-free medium also contained epithelioid colonies which were considerably larger than those in control cultures (Fig. 2C). The number of colonies, however, was less than in the untreated Ficoll prepared group. Interestingly, when these cultures were maintained for longer periods of time (up to 3 weeks), there was
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FIG. 2. Photomicrographs of control and purified cultures prepared according to the protocol outlined in Figure 1 and fixed after 8 days. Beta cells are easily identified by the presence of dark staining (aldehyde-thionin positive) cytoplasmic granules (aldehyde-thionin x 87). a. Control culture, b. Culture prepared by Ficoll gradient centrifugation. c. Culture prepared by Ficoll gradient centrifugation and subsequently treated with cystine-free medium.
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PANCREATIC BETA CELL CULTURE
641
obvious enlargement of the epithelioid TABLE 2. Insulin release into the culture medium from control and purified cultures colonies. In addition, significant numbers of mitotic figures were observed amongst Insulin released per 2-day interval the aldehyde-thionin positive cell popula(mU per culture) tion. A gradual increase in the numbers of fibroblastoid cells present between colGlucose Glucose 300 mg/100 ml 100 mg/100 ml onies was also observed during the longer(days 4-6) (days 6-8) Group term incubations. In contrast, after three weeks, control I. Control 24.4 ± 5.0 11.7 ± 5.2 cultures were heavily overgrown with a II. Ficoll 151.9 ± 32.3* 68.2 ± 20.4* dense network of fibroblastoid cells. HI. Ficoll plus Epithelioid colonies appeared multicystine-free medium 49.3 ± 9.9*t 31.4 ± 6.3* layered, so that the surface area occupied by each colony was noticeably decreased. The number of cells used to prepare each purified Longer-term cultures prepared by Ficoll culture (Groups II, III) was approximately 18 times gradient centrifugation, but not treated that for each control culture (Group I). Each value is the mean ± SEM for six experiments. with cystine-free MEM, also contained statistically significant to at least the increased numbers of fibroblastoid cells. P
necrosis of the majority of fibroblastoid cells within two days, while beta cells were considerably less affected. The resultant cultures contained an average of 72% beta cells compared to 10% in untreated control cultures. Insulin release by purified monolayers remained responsive to changes in the glucose concentration of the culture medium. (Endocrinology 96: 637, 1975)
M
ONOLAYER cultures containing pancreatic beta cells have been successfully employed to study insulin biosynthesis and release, beta cell replication, and beta cell morphology by both light and electron microscopy (1-7). Unfortunately despite recent advances in methodology for preparing such cultures from newborn rats, the beta cells still constitute only a small fraction of the total cell population. This obviously restricts their usefulness. Studies were therefore undertaken to develop means for preparing purified beta cell monolayer cultures from newborn rats. The method presented is based on Ficoll gradient centrifugation to separate endocrine from exocrine cells in the initial cell suspension (8,9), followed by incubation of the resulting cultures with a nutritionally deficient medium to eliminate the majority of residual extraneous cells. Potential applications for this method are discussed. Materials and Methods Preparation of monolayers Cell suspensions were prepared by dissociating pancreases from two to three day old rats (C-D strain, Charles River) with trypsin and collagenase as previously described (4,5). An average of 70 pancreases were used in each experiment: The cell count was adjusted to Received September 23, 1974.
7.5 x 105 viable cells per ml, and 5 ml removed to prepare a control culture. The remainder of the suspension (approximately 180 ml) was centrifuged at 150 x g for 5 min and the cell pellet resuspended in 4 ml of an ice cold solution of 25% (wt/vol) dialyzed Ficoll (9) in growth medium. Growth medium was TCM 199 containing 10% fetal calf serum, 400 U of sodium penicillin per ml, and 300 rng of glucose per 100 ml unless specified otherwise. The cell suspension was overlaid with an additional 4 ml of 20% Ficoll in growth medium and centrifuged for 10 min at 1000 x g. Preliminary experiments indicated that the portion of the gradient from the 20-25% interface to just above the cell pellet was highly enriched in beta cells, while acinar cells remained in the pellet. This fraction enriched in beta cells was removed, diluted with growth medium and centrifuged to remove the Ficoll. The final pellet was suspended in 10 ml of growth medium and divided equally between two cultures. Sixty millimeter petri dishes were used for all cultures. Incubation was at 37 C in a humidified atmosphere of 95% air: 5% CO2 as previously reported (4,5). The primary cell suspension was decanted into new dishes after 14-16 h to eliminate large numbers of fibroblastoid cells which attached to the original dishes (3-5). The following day the control and one of the Ficoll-treated cultures were washed with growth medium and reincubated. The other Ficoll-treated culture was washed and reincubated with cystine-free Eagle's Minimal Essential Medium (Grand Island Biological Company Select-amine Kit) containing 10% dialyzed fetal calf serum, 300 mg of glucose per 100 ml, and
637
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Endo • 1975 Vol 96 • No 3
CHICK, LIKE AND LAURIS
638
400 U of penicillin per ml. Incubation with the cystine-free medium led to rapid necrosis of the overwhelming majority of residual fibroblastoid cells while significant numbers of endocrine cells survived this treatment. After 2 days, all three types of cultures were again washed and reincubated with growth medium for two additional days. Finally, during the last 2 days of the experiment the glucose concentration in the growth medium was lowered to 100 mg/100 ml to permit regranulation of the beta cells and thus facilitate identification following staining with aldehyde-thionin (Fig. 1). In addition, in three experiments the effects of longer-term incubation were evaluated by maintaining cultures for a total of 3 weeks rather than 8 days. The protocol followed was similar to that already described with the exception that the glucose concentration in the medium was lowered from 300 to 100 mg/100 ml during the 2 days prior to fixation and staining rather than during days 6 to 8.
face of each culture dish. Cells in each field were assigned to one of three categories depending upon their shape and staining characteristics: 1. Fibroblastoid; 2. Epithelioid, aldehyde-thionin positive (beta cells); 3. Epithelioid, aldehyde-thionin negative.
Results
Morphology Gradient separation. Four layer Ficoll gradients (25%, 23%, 2Q%, 11%) similar to those used to isolate islets from adult rats were initially employed to develop the two layer gradient used in these experiments (8,9). Using this simpler gradient, the
majority of cells remained at the bottom of the 25% Ficoll layer. The predominant cell type in this pellet, as evaluated by phase contrast microscopy (4,5,7), consisted of acinar cells. Cultures prepared from this portion of the gradient contained some beta Insulin release cells, as evidenced by aldehyde-thionin Aliquots of medium (Fig. 1) were assayed for immunoreactive insulin (IRI) content as previ- staining. These presumably had formed ously described, using purified rat insulin stand- aggregates with the denser acinar cells and sedimented in the gradient with them (7). ards (4,5). Cultures prepared from the upper portion of the 20% Ficoll layer contained relatively Light microscopy and morphometric analysis few cells. There were rare aldehyde-thionin Living cultures were examined periodically positive (beta) cells in addition to fibroblasby phase contrast microscopy. After 8 days of toid appearing cells. The portion of the incubation they were fixed and stained with gradient from the 20-25% interface to just aldehyde-thionin for identification of beta cells above the pellet at the bottom of the (3-5). Because of rapid fading of stained fibroblastoid cells, morphometric studies were per- gradient yielded cultures enriched in formed immediately following staining. A aldehyde-thionin positive (beta) cells, but minimum of 1000 cells were counted in ran- also containing significant numbers of domly selected fields distributed over the sur- fibroblastoid cells. The majority of these fibroblastoid cells attached to the original Plonl Sove Medium Sove Medium dishes during the initial 14-16 h of incubaDecora for IRI forlRI Cultures and were therefore discarded when tion I I the cell suspension was decanted into new Doys : 0 e dishes (3-5). Medium : 199 199 199 199or cystine-free MEM
Glucose KX>mg/)0Onl (Regroiiulotion F^riod)
FIG. 1. Protocol followed in preparing and studying control and purified cultures. The glucose concentration in the culture medium was 300 mg/100 ml except during days 6-8 when this was lowered to 100 mg/ 100 ml to permit beta cell regranulation prior to staining.
Cystine-free medium. The use of cystinefree medium was based on our observation that incubation of rat pancreatic monolayer cultures with 100% fetal calf serum resulted in obvious necrosis of the majority of fibroblastoid cells within 1-2 days. Under
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2014. at 02:33 For personal use only. No other uses without permission. . All rights reserved.
PANCREATIC BETA CELL CULTURE these same conditions the beta cells in these cultures were affected to a considerably lesser extent. Subsequent amino acid analysis of the fetal calf serum used in these experiments revealed that of the amino acids reported to be essential for most mammalian cells in culture (10) all were present in adequate amounts with the exception of cystine. The half-cystine content of the fetal calf serum was only 2 /x,mol per liter. The necrosis of fibroblastoid cells in the cultures observed under phase contrast microscopy could be prevented by the addition of cystine (200 /xmol per liter) to the fetal calf serum (11). Finally, the effects of 100% fetal calf serum without added cystine were duplicated by the use of cystine-free Eagle's Minimal Essential Medium (MEM). As anticipated (6), complete MEM exerted no discernible morphologic effects. . Although the cultures in the majority of experiments included in this report were incubated for a total of 2 days with cystinefree MEM, smaller numbers of experiments were also conducted in which incubation was continued for a total of 5 days. Although fibroblastoid cells were almost totally eliminated after this longer incubation with the nutritionally deficient medium, the numbers of endocrine cells surviving were very markedly reduced. From these experiments it was concluded that a 2-3-day incubation with the cystinefree medium was optimal in terms of preserving significant numbers of beta cells while still achieving relatively high purity through elimination of extraneous cell types. Although cystine is an essential amino acid for most mammalian cells in culture, it is not an essential amino acid for the whole animal, since it is synthesized in the liver from the essential amino acid methionine and the nonessential amino acid L-serine (12). In view of the possibility that similar conversion pathways were also present to a limited extent in beta cells, cultures were incubated in cystine-free MEM containing ten-fold increased levels
639
of L-methionine and to which the nonessential amino acid L-serine was also added (42 mg per liter). Under phase contrast, cultures incubated with this medium were indistinguishable from those incubated in cystine-free medium without the added amino acids. Since tyrosine is a second amino acid essential for most mammalian cells in culture, but not for the whole animal (10), the effects of tyrosine-free MEM were also evaluated. Interestingly, after 3 days of incubation, no obvious effects could be discerned by phase microscopy on either fibroblastoid or epithelioid cells. The construction of these deficient media was facilitated by the availability commercially of a kit designed to permit deletion of specific amino acids in MEM. Qualitative evaluation of the three types of cultures. As previously reported (4), control cultures contained numerous scattered col-' onies of epithelioid cells surrounded by a dense network of fibroblastoid cells. The majority of these epithelioid cells contained aldehyde-thionin positive granules (Fig. 2A). In contrast, cultures prepared by Ficoll gradient separation contained considerably greater numbers of these epithelioid colonies. In addition, the colonies were noticeably larger in size than in the control group. The majority of these epithelioid cells were also aldehydethionin positive (Fig. 2B). Although fibroblastoid cells occupied the spaces between the epithelioid colonies, their numbers were limited by the large amount of space taken up by the latter. Finally, cultures prepared by Ficoll gradient separation and then treated with cystine-free medium also contained epithelioid colonies which were considerably larger than those in control cultures (Fig. 2C). The number of colonies, however, was less than in the untreated Ficoll prepared group. Interestingly, when these cultures were maintained for longer periods of time (up to 3 weeks), there was
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FIG. 2. Photomicrographs of control and purified cultures prepared according to the protocol outlined in Figure 1 and fixed after 8 days. Beta cells are easily identified by the presence of dark staining (aldehyde-thionin positive) cytoplasmic granules (aldehyde-thionin x 87). a. Control culture, b. Culture prepared by Ficoll gradient centrifugation. c. Culture prepared by Ficoll gradient centrifugation and subsequently treated with cystine-free medium.
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PANCREATIC BETA CELL CULTURE
641
obvious enlargement of the epithelioid TABLE 2. Insulin release into the culture medium from control and purified cultures colonies. In addition, significant numbers of mitotic figures were observed amongst Insulin released per 2-day interval the aldehyde-thionin positive cell popula(mU per culture) tion. A gradual increase in the numbers of fibroblastoid cells present between colGlucose Glucose 300 mg/100 ml 100 mg/100 ml onies was also observed during the longer(days 4-6) (days 6-8) Group term incubations. In contrast, after three weeks, control I. Control 24.4 ± 5.0 11.7 ± 5.2 cultures were heavily overgrown with a II. Ficoll 151.9 ± 32.3* 68.2 ± 20.4* dense network of fibroblastoid cells. HI. Ficoll plus Epithelioid colonies appeared multicystine-free medium 49.3 ± 9.9*t 31.4 ± 6.3* layered, so that the surface area occupied by each colony was noticeably decreased. The number of cells used to prepare each purified Longer-term cultures prepared by Ficoll culture (Groups II, III) was approximately 18 times gradient centrifugation, but not treated that for each control culture (Group I). Each value is the mean ± SEM for six experiments. with cystine-free MEM, also contained statistically significant to at least the increased numbers of fibroblastoid cells. P
