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Insulin secretory on isolated pig islets
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3.27
Cell Transplantation • Volume 5, Number 5S-2, 1996 LARGE SCALE HEPATOCYTE CULTURE FOR ARTIFICIAl. LIVER SUPPORT. Nnik S, Santangini HA, Treulder DM Mullon CJP, Jauregai HO; Providence, USA.
3.28
Microcarrier attached bepatecytes have uppfications both i~l artificial liver support and cell transplantation therapies. In either scenario, the maintenance o f / n vivo metabolic functions are of primary importance. Previous studies of anchorage dependent cells in vitro have suggested a correlation between 3 dimensional morphology and the maintenance of function. In this study, aggregate formation, benzodiazepine metabolisn~ and cell proliferation by porcine hepatocytes cultured on different micrncarrier beads in roller bottles were compared. Porcine hepatocytes were seeded on microcarriers at cell density normalized to surface area by adjustment of bead number (6.25 cm2/l x 10e cells) and maintained In roller bottle culture for 6 days. Microcarrier sizes ranged from 90-1,000 tun Mean diazepam metabolite production ranged from 18-31 IJ.g/l x 10e cells, with overall highest function and DNA content measured on culture day 4. Ratios based on aggregate size (#t of microcarriers) were similar for most microcarriers, but th~ aggregation characteristics of one type were much higheri Contrary to the reports of others, the increased cell contact ano tissue- like structure observed in those cultures did not result in improved cell proliferation or metabolic capacity as measured by DNA content or diazepam metabolite production, respectively. In conclusion, roller bottle microcarrier cultur0 allows large numbers of cells to be maintained with greatee ease than monolayer cultures, and supports mass production cell products, but does not improve individual cell function in vitro.
3.29
THE EFFECT OF DONOR AGE ON PORCINE INSULIN SECRETION. Jay Tit, Heald KA, Downing R; Worcester UK, Pig islets have potential for use in clinical xenotrausplantation as a treatment for diabetes mellitus. To investigate the effect of the age of donor pancreas on insulin secretion we have studied isolated islets isolated from 5, 12 and 24 week old pigs (n=8 per group). Isolated islets were incubated with secretagogues for 60 minutes at 37°C and insulin measured by radioimmnnoassay. Insulin secretion was expressed as a stimulation index and compared using the Mann-Whitney U test. Values are expressed as mean ± SD in age order, i.e, 5, 12, 24 weeks. Stimulation indices in response to 15mmol 1-~ glucose were not significantly different between the age groups (1.1 ± 0.2, 1.3 + 0.2 and 1.1 + 0.1), but stimulation indices in response to 15retool 1"~ glucose + 10retool I"l thcophylline, and 10mmol !q isoprenaline were significantly greater fi'om 5 and 12 week old compared to 24 week old pigs (Glucose and theophylline: 6.9 ± 3.9 and 4.7 ± 2.1 vs 1.6 ± 0.5; isoprenaline: 4.4 ± 2.6 and 3.1 ± 1.2 vs 1.9 + 0.6). Islets from 5 week old pigs gave the greatest insulin secretory response to 15retool !-~ glucose + 0.05mmol 1-1 carbachol, 15retool 1"~mannose, mixed amino acids, 20mmol 1-~ leucine and 0.1mmol 1-1 tulbutamide, but values were not significantly different from those obtained from 12 and 24 week old pigs. In conclusion, donor age had a significant effect upon insulin secretory respnnses to glucose + theophylline and isoprenaline.
INSULIN SECRETORY STUDIES ON ISOLATED PIG ISLETS Heald KA, Jay TR, Downing R; Worcester UK The response of isolated pig islets to stimulation with glucose in-vitro is unpredictable. The aim of the study was to determine if the response to glucose is influenced by culture conditions. Islets were isolated from 5 week old pigs by manual collagenase digestion and purified using Histopaque gradients. All preparations were cultured in CMRL containing 20% pig serum for 2 days. Insulin secretion in response to 15mM glucose (G) and 15mM glucose + 10ram theophylline (GT) was determined by static challenge. Stimulation indices were expressed as mean ± SD and compared using Mann-Whitney U-test. Insulin secretory response was unaffected by purification, [non-porified (G: 1.2 ± 0.3; GT: 3.7 ± 3.1) vs purified (G: 1.3 + 0.3; GT: 6.2 ± 4.3)] temperature [26°C (G: 1.1 ± 0.3; GT: 5.9 ± 4.8) vs 37°C (G: 1.3 ± 0.2; GT: 6.3 ± 4.4] or time in culture [2 days (G: 1.2 ± 0.3; GT: 4.9 ± 3.9) vs 7 days (G: 1.2 ± 0.3; GT: 4.0 ± 3. ]. Responses from flee islets and islets encapsulated in calcium alginate on the day of isolation were not significantly different, but ff islets were encapsulated immediately before challenge, secretion was significantly enhanced [free islets (G: 1.3 ± 0.2;GT:6.3 ± 5.0) vs freshly encapsulated islets (G: 2.3 ± 1.4; GT: 18.5 ± 2.7 (p<0.05). In conclusion, none of the culture conditions tested significantly improved glucose induced insulin secretion. However, encapsulation of islets immediately before challenge resulted in a significantly increased insulin response.
3.30
A HISTOLOGICAL STUDY OF ~-CELLS IN PORCINE PANCREAS AGED 5, 12 & 24 WEEKS. Jay TR, Heald KA, Downing R; Worcester UK. Islet transplantation is a potential treatment for diabetes mellitus and porcine pancreata may provide a readily available source of islets. The size, number and distribution of islets in the pancreas may influence the choice of age of donor for xenotransplantation. Samples from the dorsal and ventral pancreas of 5, 12 and 24 week old hybrid pigs (Cotswold 12X) were fixed in formal saline, processed in paraffin wax and stained with an avidin/biotin immunohistochemieal kit for insulin. Mean diameter of 13-coll groups and lone 13.-coils, were measured in 1ram2 grid areas (n=10) and collated into groups according to size. Size distribution (%) and percentage ~-.cell volume density relative to non-insulin staining tissue were calculated. Islets in 5 week old pancreata were smaller than those in 12 and 24 week old pancreata. Median (range) diameter of ~-cell groups (~tm) were: 5 week - dorsal 10 (10o90), ventral 10 (10-95); 12 week dorsal 25 (10-200), ventral 30 (10-180) and 24 week dorsal 22 (10-200), ventral 15 (10-100). The [~-cell volume density expressed as a pecentage of total pancreas volume was: 5 week - dorsal 0.91%, ventral 0.95%; 12 week - dorsal 2.97%, ventral 2.99% and 24 week - dorsal 2.27%, ventral 0.79°/0. This study shows that the average islet diameter and 13-cell volume density relative to non-insulin staining tissue increased between the ages of 5 and 12 weeks, but decreased during the period 12- 24 weeks. These differences may have implications on the isolation and function of transplanted islets if young pigs are to be used as donors. -
3.27
Cell Transplantation • Volume 5, Number 5S-2, 1996 LARGE SCALE HEPATOCYTE CULTURE FOR ARTIFICIAl. LIVER SUPPORT. Nnik S, Santangini HA, Treulder DM Mullon CJP, Jauregai HO; Providence, USA.
3.28
Microcarrier attached bepatecytes have uppfications both i~l artificial liver support and cell transplantation therapies. In either scenario, the maintenance o f / n vivo metabolic functions are of primary importance. Previous studies of anchorage dependent cells in vitro have suggested a correlation between 3 dimensional morphology and the maintenance of function. In this study, aggregate formation, benzodiazepine metabolisn~ and cell proliferation by porcine hepatocytes cultured on different micrncarrier beads in roller bottles were compared. Porcine hepatocytes were seeded on microcarriers at cell density normalized to surface area by adjustment of bead number (6.25 cm2/l x 10e cells) and maintained In roller bottle culture for 6 days. Microcarrier sizes ranged from 90-1,000 tun Mean diazepam metabolite production ranged from 18-31 IJ.g/l x 10e cells, with overall highest function and DNA content measured on culture day 4. Ratios based on aggregate size (#t of microcarriers) were similar for most microcarriers, but th~ aggregation characteristics of one type were much higheri Contrary to the reports of others, the increased cell contact ano tissue- like structure observed in those cultures did not result in improved cell proliferation or metabolic capacity as measured by DNA content or diazepam metabolite production, respectively. In conclusion, roller bottle microcarrier cultur0 allows large numbers of cells to be maintained with greatee ease than monolayer cultures, and supports mass production cell products, but does not improve individual cell function in vitro.
3.29
THE EFFECT OF DONOR AGE ON PORCINE INSULIN SECRETION. Jay Tit, Heald KA, Downing R; Worcester UK, Pig islets have potential for use in clinical xenotrausplantation as a treatment for diabetes mellitus. To investigate the effect of the age of donor pancreas on insulin secretion we have studied isolated islets isolated from 5, 12 and 24 week old pigs (n=8 per group). Isolated islets were incubated with secretagogues for 60 minutes at 37°C and insulin measured by radioimmnnoassay. Insulin secretion was expressed as a stimulation index and compared using the Mann-Whitney U test. Values are expressed as mean ± SD in age order, i.e, 5, 12, 24 weeks. Stimulation indices in response to 15mmol 1-~ glucose were not significantly different between the age groups (1.1 ± 0.2, 1.3 + 0.2 and 1.1 + 0.1), but stimulation indices in response to 15retool 1"~ glucose + 10retool I"l thcophylline, and 10mmol !q isoprenaline were significantly greater fi'om 5 and 12 week old compared to 24 week old pigs (Glucose and theophylline: 6.9 ± 3.9 and 4.7 ± 2.1 vs 1.6 ± 0.5; isoprenaline: 4.4 ± 2.6 and 3.1 ± 1.2 vs 1.9 + 0.6). Islets from 5 week old pigs gave the greatest insulin secretory response to 15retool !-~ glucose + 0.05mmol 1-1 carbachol, 15retool 1"~mannose, mixed amino acids, 20mmol 1-~ leucine and 0.1mmol 1-1 tulbutamide, but values were not significantly different from those obtained from 12 and 24 week old pigs. In conclusion, donor age had a significant effect upon insulin secretory respnnses to glucose + theophylline and isoprenaline.
INSULIN SECRETORY STUDIES ON ISOLATED PIG ISLETS Heald KA, Jay TR, Downing R; Worcester UK The response of isolated pig islets to stimulation with glucose in-vitro is unpredictable. The aim of the study was to determine if the response to glucose is influenced by culture conditions. Islets were isolated from 5 week old pigs by manual collagenase digestion and purified using Histopaque gradients. All preparations were cultured in CMRL containing 20% pig serum for 2 days. Insulin secretion in response to 15mM glucose (G) and 15mM glucose + 10ram theophylline (GT) was determined by static challenge. Stimulation indices were expressed as mean ± SD and compared using Mann-Whitney U-test. Insulin secretory response was unaffected by purification, [non-porified (G: 1.2 ± 0.3; GT: 3.7 ± 3.1) vs purified (G: 1.3 + 0.3; GT: 6.2 ± 4.3)] temperature [26°C (G: 1.1 ± 0.3; GT: 5.9 ± 4.8) vs 37°C (G: 1.3 ± 0.2; GT: 6.3 ± 4.4] or time in culture [2 days (G: 1.2 ± 0.3; GT: 4.9 ± 3.9) vs 7 days (G: 1.2 ± 0.3; GT: 4.0 ± 3. ]. Responses from flee islets and islets encapsulated in calcium alginate on the day of isolation were not significantly different, but ff islets were encapsulated immediately before challenge, secretion was significantly enhanced [free islets (G: 1.3 ± 0.2;GT:6.3 ± 5.0) vs freshly encapsulated islets (G: 2.3 ± 1.4; GT: 18.5 ± 2.7 (p<0.05). In conclusion, none of the culture conditions tested significantly improved glucose induced insulin secretion. However, encapsulation of islets immediately before challenge resulted in a significantly increased insulin response.
3.30
A HISTOLOGICAL STUDY OF ~-CELLS IN PORCINE PANCREAS AGED 5, 12 & 24 WEEKS. Jay TR, Heald KA, Downing R; Worcester UK. Islet transplantation is a potential treatment for diabetes mellitus and porcine pancreata may provide a readily available source of islets. The size, number and distribution of islets in the pancreas may influence the choice of age of donor for xenotransplantation. Samples from the dorsal and ventral pancreas of 5, 12 and 24 week old hybrid pigs (Cotswold 12X) were fixed in formal saline, processed in paraffin wax and stained with an avidin/biotin immunohistochemieal kit for insulin. Mean diameter of 13-coll groups and lone 13.-coils, were measured in 1ram2 grid areas (n=10) and collated into groups according to size. Size distribution (%) and percentage ~-.cell volume density relative to non-insulin staining tissue were calculated. Islets in 5 week old pancreata were smaller than those in 12 and 24 week old pancreata. Median (range) diameter of ~-cell groups (~tm) were: 5 week - dorsal 10 (10o90), ventral 10 (10-95); 12 week dorsal 25 (10-200), ventral 30 (10-180) and 24 week dorsal 22 (10-200), ventral 15 (10-100). The [~-cell volume density expressed as a pecentage of total pancreas volume was: 5 week - dorsal 0.91%, ventral 0.95%; 12 week - dorsal 2.97%, ventral 2.99% and 24 week - dorsal 2.27%, ventral 0.79°/0. This study shows that the average islet diameter and 13-cell volume density relative to non-insulin staining tissue increased between the ages of 5 and 12 weeks, but decreased during the period 12- 24 weeks. These differences may have implications on the isolation and function of transplanted islets if young pigs are to be used as donors. -