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Biomedical subjects

M D Brendel

Publications and source records attributed to M D Brendel.

47 records · Page 3Linked to original sources
PubMed1993

Successful cryopreservation of fetal porcine proislets.

This study reports the cryopreservation of purified fetal pig proislets (FPP) which were isolated by a culture technique. The FPP were equilibrated with 2 M dimethyl sulfoxide (Me2SO), cooled at a rate of 0.25 degrees C/min to -40 degrees C, transferred to liquid nitrogen (-196 degrees C), stored for 2 h to 2 months at -196 degrees C, and thawed in a 37 degrees C water bath. The morphology of frozen-thawed FPP was similar to that of noncryopreserved FPP. One hundred percent recovery of response capacity was achieved when tested with 16.7 mM glucose plus 10 mM theophylline. We have recently shown that streptozotocin is not toxic to FPP. Accordingly, we transplanted a standardized dose (10-15 mg/mouse) of either cultured of frozen-thawed FPP beneath the renal capsule of nude mice. Three to four weeks later the recipients of the FPP were rendered diabetic by iv streptozotocin. All of the mice transplanted with cultured FPP and seven of eight mice receiving cryopreserved FPP achieved normoglycemia, 74.8 +/- 32.9 and 54.7 +/- 8.1 days (P > 0.05) after transplantation. An intraperitoneal glucose tolerance test in the mice of both groups showed a flatter response to glucose compared to those of normal controls. Grafts removed from the mice with normoglycemia for > 3 weeks, in both groups, had the capacity to secrete insulin in response to 16.7 mM glucose alone and 16.7 mM glucose plus 10 mM theophylline during in vitro perifusion. Histological examination revealed that the extent of differentiation and development, in vivo of cryopreserved FPP was comparable with that of cultured FPP. These data indicate that cryopreservation, with the protocol used here, is successful in maintaining functional viability of frozen-thawed FPP. This study is valuable for clinical islet transplantation research.

Animals↗
PubMed1991

Persistent reversal of diabetes by transplantation of fetal pig proislets into nude mice.

Facing the limited availability of human adult and fetal pancreases, fetal pig proislets (pancreatic islet precursors) were investigated in view of several inherent advantages. Six litters of fetuses of mean +/- SE gestational age 75 +/- 3 days were obtained from commercially available farm pigs. Pancreatic tissue was gently digested with collagenase, then a 10-day culture was performed. During culture, fetal proislets showed no insulin response to glucose alone but a significant response to glucose plus theophylline. The insulin content per microgram of DNA in the cultured proislets continuously increased. Histological examination by immunoperoxidase staining showed that, apart from single insulin- and glucagon-positive cells, there were no discrete islets in the pancreatic tissue and the cultured proislets. Diabetes was induced with streptozocin (STZ) in eight nude mice 3-4 wk after proislet transplantation and in another eight nude mice without transplantation. During the initial week, blood glucose levels of mice in both groups increased rapidly. The mean +/- SE peak value of blood glucose levels in the transplanted group was 20.4 +/- 2.0 mM and was 20.1 +/- 1.3 mM in the group without transplantation. Simultaneously, body weight decreased from 29.5 +/- 0.7 to 21.5 +/- 0.9 g and from 27.9 +/- 0.7 to 19 +/- 1 g in the groups, respectively. Afterward, blood glucose levels of mice in the transplanted group gradually decreased, and normoglycemia was achieved in all mice within 50 +/- 13 days after injection of STZ, i.e., 74 +/- 13 days after transplantation. The group without transplantation persistently maintained blood glucose levels greater than 16.7 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Percutaneous transhepatic catheterization of the portal vein: A combined CT- and fluoroscopy-guided technique.

Combined CT- and fluoroscopy-guided transhepatic portal vein catheterization was performed in 44 patients selected for pancreatic islet cell transplantation. The method allowed catheterization with a single puncture attempt in 39 patients. In four patients two attempts and in one patient four attempts were necessary. One minor hematoma of the liver capsule occurred that required no further treatment. Compared with other methods the average number of puncture attempts was reduced.

Adult↗
PubMedDate not supplied

Adaption of neutral protease activity for islet isolation from the long-term two-layer method-stored pig pancreas.

BACKGROUND: Islet release from the pancreas is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. To prove the hypothesis that adjustment of NP reduces islet damage after prolonged ischemia, adult pig pancreata were digested after 7-hour preservation by the two-layer method (TLM) using a 2-component enzyme blend consisting of collagenase NB-8 and NP. METHODS: After intraductal University of Wisconsin (UW) flush resected pancreata were distended with 4.4 PZ-U/g of UW-dissolved Serva collagenase either before (TLM-preloaded, n = 7) or after (TLM-postloaded, n = 10) cold storage, or for immediate processing (n = 6). NP was adjusted after preliminary experiments to respectively 1.1, 0.2, or 0.8 DMC-U/g for unstored, TLM-preloaded, or postloaded organs. RESULTS: Purified islet yield decreased from 3670 +/- 730 islet equivalents (IEQ)/g in unstored pancreata to 1800 +/- 180 and 2080 +/- 290 IEQ/g in TLM-preloaded or postloaded organs, respectively (P < .05). Although purity was always >90%, IEQ recovery was significantly decreased in TLM-preloaded pancreata. Quality control revealed consistently high viability as determined using trypan-blue exclusion (>95%) or formazan production. Compared with unstored organs (2.47 +/- 0.36; P < .05), glucose stimulation index was reduced in TLM-preloaded (1.48 +/- 0.15) and TLM-postloaded pancreata (1.81 +/- 0.20). Normoglycemia in diabetic nude mice transplanted with islets from TLM-preloaded pancreata was transient in contrast to sustained function in the other groups. CONCLUSIONS: Significant amounts of viable pig islets can be isolated after prolonged TLM preservation by reducing NP activity. Nevertheless, early enzyme administration prior to long-term storage deteriorates islet graft function.

Adenosine↗
PubMedDate not supplied

Long-term preservation of the pig pancreas by a one-layer method for successful islet isolation.

BACKGROUND: Pancreas preservation by two-layer method (TLM) was recently established for clinical islet transplantation. The extensive use of TLM would require enormous efforts to solve logistical and technical problems. Omitting University of Wisconsin solution (UW) as second layer would facilitate the regular application of oxygenated perfluorocarbon; (PFC). To clarify whether long-term pancreas preservation is feasible by this simplified procedure, pancreases from retired breeder pigs were subjected to 7-hour preservation utilizing PFC alone in a one-layer method (OLM, n = 8) or in combination with UW (TLM, n = 10). METHODS: Resected pancreata were intraductally flushed with cold UW. Subsequently, pancreata were promptly processed (n = 6) as previously described or stored by TLM or OLM. RESULTS: Compared to unstored (429200 +/- 86700 IEQ) and OLM-stored pancreases (338600 +/- 42100 IEQ), (P = ns vs unstored) postpurification islet yield decreased after TLM storage (238000 +/- 26600 IEQ, P < .05). No significant differences were found regarding purity (>90%), adenosine triphosphate (ATP) content, and viability as determined by formazan production and trypan-blue exclusion (>95%). Glucose stimulation index of freshly isolated islets (2.5 +/- 0.4) was significantly decreased after TLM storage (1.8 +/- 0.2, P < .05) but not after OLM storage (2.3 +/- 0.6). Islet transplantation in diabetic nude mice demonstrated sustained graft function in all experimental groups. CONCLUSIONS: This study demonstrates that viable pig islets can be successfully isolated after prolonged ischemia utilizing PFC alone for oxygenation of cold-stored pig pancreases. The easy handling of OLM could facilitate the regular application of PFC as pancreas preservation solution.

Adenosine↗
PubMedDate not supplied

Influence of neutral protease activity on human islet isolation outcome.

UNLABELLED: Observations in rat pancreata have revealed that enzymatic islet release is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. Since no information is available about the effect of NP activity on islet release from the human pancreas, the present study evaluated the effect of various NP concentrations on the outcome of human islet isolation. METHODS: Following intraductal collagenase distension, pancreata obtained from adult multiorgan donors were digested using 2000 PZ-U of purified Serva collagenase NB 1 supplemented with 2.6 (n = 10) or 4.5% (DMC-U/PZ-U) (n = 10) of NP. RESULTS: Increasing NP from 2.6% to 4.5% reduced the amount of undigested tissue from 22 +/- 2 to 17 +/- 2 g (P < .05) while simultaneously increasing the volume of digested tissue (26 +/- 2 vs 40 +/- 3 mL, P < .01). Increased NP concentrations increased the islet yield prepurification (459,800 +/- 22,900 vs 587,600 +/- 69,000 IEQ, P < .05), but simultaneously affected islet purification, resulting in equal islet yields (345,700 +/- 31,200 vs 391,500 +/- 35,400 IEQ, NS) and less purity (70 +/- 6 vs 49% +/- 5%, P < .01). A NP concentration of 4.5% reduced the stimulation index (4.7 +/- 1.2 vs 2.0 +/- 0.5, P < .01) and viability (100 +/- 1 vs 95% +/- 3%, P < .05). CONCLUSIONS: Although increased NP activity seems to improve islet release from adult human pancreata, it significantly affects islet viability and function. The reduction in purity reflected damage to acinar tissue by increased NP activity presumably affecting islet integrity.

Adult↗
PubMedDate not supplied

A minimally invasive technique for intrathymic cell transplantation in the dog.

As an alternative to drug immunosuppression, attempts at inducing donor-specific tolerance by intrathymic (IT) inoculations to transplant recipient of donor origin alloantigenic products has proven very promising. Using fiber optic thoracoscopy, a technique for the study of this phenomena was developed for the dog. We show an approach to the dog thymus using fiber optics for injection of bone marrow (BM) cells as the tolerogen. Bone marrow was retrieved from the donor beagles and purified using an automated Ficoll-Paque gradient technique. The purified cellular suspension was injected into the thymus through a small intercostal incision with the use of an injection needle port guided by the use of a rigid fiberoptic scope. To demonstrate engraftment, supravital staining with Fluorescein Diacetate of the BM cells was performed prior to inoculation. Immunofluorescence of cryostat sections obtained at necropsy confirmed the presence of viable BM cells up to several days after transplantation. Results of this study show that the thoracoscopic approach to the thymus can be safely and effectively used for IT inoculation studies in dogs.

Animals↗
PubMedDate not supplied

Improved functional survival of human islets of Langerhans in three-dimensional matrix culture.

The current study evaluates functional survival of human islets maintained in tissue culture for up to 4 wk in suspension media (CMRL-1066 with supplements) and contrasts these results with immobilizing three-dimensional matrices (agarose or alginate). The absolute number and volume of islets retrieved from agarose is significantly higher after two and four wk of culture compared to conventional free-floating media. In vitro function of islets, assessed by insulin/DNA content, insulin secretion into the culture media over 24 h and glucose-theophylline stimulated insulin release in a dynamic perifusion system, was not significantly different between free-floating and matrix preserved islets. In vivo islet function was evaluated by the effectiveness for reversal of insulin-dependent diabetes mellitus by transplantation of the islets under the kidney capsule of nude mice. Although adequate insulin responses to glucose were seen after culture in conventional or matrix media, only agarose embedded islets were consistently able to induce normoglycemia in diabetic recipients after 14 days of culture. Additional transplantation experiments defined the threshold level required to reverse diabetes to be between 1,000 and 1,500 agarose preserved islets. Our data suggest improved engraftment of human islets after agarose culture. This culture method may be of benefit for the accumulation of functionally competent human islets, thus facilitating the implementation of clinical protocols that utilize freshly isolated islets from multiple donors without the need for cryopreservation.

Alginates↗
PubMedDate not supplied

Assessment of intracellular insulin content during all steps of human islet isolation procedure.

This study investigated the recovery of pancreatic insulin content during human islet isolation prior to and after digestion-filtration, continuous Hanks-Ficoll gradient purification (n = 20), and 3-4 day culture at 22 degrees C (n = 6). The native insulin content varied in a wide range from 28.4 U to 360.8 U/pancreas. After digestion the initially measured average insulin content of 115.8 +/- 20.8 U/pancreas (mean +/- SEM) increased to 264.6 +/- 22.8% (p < 0.001). This increase of insulin during pancreas digestion was attributed to the asymetrical distribution of insulin within the pancreas. Sampling of insulin within the pancreatic caput seemed not to be representative for the insulin content of the complete native organ, because the ratio of insulin per gram tissue within the pancreatic cauda compared to the caput (n = 5) was 2.4 +/- 0.4 (p < 0.05). After purification total insulin recovery was 55.3 +/- 4.8% (p < 0.001). Because recovery of islet equivalent number (IEQ) (83.7 +/- 4.4%) exceeded insulin recovery, insulin/IEQ ratio decreased from 656.8 +/- 70.6 microU/IEQ before purification to 436.4 +/- 58.1 microU/IEQ (p < 0.001) after purification. After 22 degrees C culture (n = 6) recovery of insulin and IEQ was 80.1 +/- 8.1% (p < 0.05) and 92.8 +/- 3.5% (p = NS), respectively. Insulin content per IEQ decreased to 85.8 +/- 6.5% (p < 0.05). This study clearly shows that most of islet insulin is lost during purification. This seems to be caused rather by an amplified insulin release than by the loss of islets itself. This release may facilitate the separation of endocrine and exocrine tissue by gradient centrifugation, but may also accelerate islet exhaustion detrimental for long-term insulin independence.

Cell Separation↗